J. Phy8iol. (1979), 287, pp. 45-56 With 3 text-ftgurew Printed in Great Britain
45
CATION TRANSPORT ACROSS THE GUINEA-PIG PLACENTA PERFUSED IN SITU
BY D. J. BAILEY, M. W. B. BRADBURY, VENETIA M. FRANCE, RUTH HEDLEY, SAROJ NAIK AND HELEN PARRY From the Department of Physiology, King's College, Strand, London WC2R 2LS
(Received 30 January 1978) SUMMARY
1. The guinea-pig placenta perfused in situ via the umbilical circulation has been used to measure unidirectional fluxes of Na from mother to fetus, and in the reverse direction, with 24Na and 22Na. There was no significant difference between the two fluxes, each being 22 /tmole. min-. 2. Ouabain 10-5 M in the perfusion fluid had no detectable effect on radioisotopic movement of Na in either direction. 3. Unidirectional fluxes of 42K in both directions were approximately equal at 1*7 ,umole.min-1 mother to fetus and 18 jumole.min-1 in the reverse direction, despite a K concentration of 3-4 mm on the maternal side and 5 0 mm on the fetal side of the placenta. 4. Extraction of 42K and 86Rb from the perfusion fluid was inhibited by 43 % by 10-5 M-ouabain in the fluid. This effect was largely due to a reduction of isotope uptake by the placental tissue. 5. The relative permeabilities of the placenta, mother to fetus, were Rb K (3.2) > Na (1.0) > Li (0.55). 6. Under the experimental conditions, the electrical potential difference between perfusion fluid and maternal blood was 6 mV (fetus negative). It was shifted towards the positive by a low Na fluid. 7. The results suggest the presence of a dominant Na-K pump (active component towards mother) sited at the maternal-facing membrane of the syncytiotrophoblast together with a subsidiary pump oriented in the opposite direction and probably sited at the fetal-facing membrane of the syncytiotrophoblast. 8. A high proportion of Na movement particularly towards the fetus is probably passive, occurring through water-filled spaces, whilst K movement is more dependent on active transport. INTRODUCTION
Mechanisms controlling fetal nutrition and homoeostasis are at present little understood. However, the placenta is generally regarded as being the organ responsible for such control. Early studies demonstrated the presence of a transplacental potential difference (p.d.) (Meschia, Wolkoff & Barron, 1958; Widdas, 1961; Mellor, 1969; of fetal K homoeostasis (Stewart & Welt, 1961; Serrano, Talbert & Welt, 1964)
D. J. BAILEY AND OTHERS and Ca homoeostasis (Bawden & Wolkoff, 1967; Greeson, Crawford, Chandler & Bawden, 1968; Bradbury, Crowder, Desai, Reynolds, Reynolds & Saunders, 1972). Placental Na transport has been studied more recently in the guinea-pig (8tulc & 8vihovec, 1973; 1977), indicating some evidence of active transport from fetus to mother, although earlier studies (Schr6der, Stolp & Leichtweiss, 1972) in the same preparation failed to find conclusive evidence for an active process. The present studies were undertaken to obtain basic facts concerning the transport of four monovalent cations across the guinea-pig placenta. The results have allowed construction of a model consistent with our present understanding of electrolyte exchange. Such a model is an essential preliminary to full characterization of the homoeostatic mechanism for ions. A preliminary account of this work has been given before the Physiological Society (Bradbury, France, Hedley & Parry, 1977). 46
METHODS
Preparation of the pregnant guinea-pig. Pregnant guinea-pigs at 59-61 days' gestation were delivered by British Rail from Redfern Animal Breeders Ltd., Brenchley, Kent, from 1 to 5 days before the experiment. The weighed guinea-pig was gently restrained while lignocaine (Lidothane) was injected s.c. into the lower forelimb. A dilute solution of pentobarbitone (10 mg/ml.) was slowly infused into the exposed brachial vein over a period of 15 min. The normal dose was 25 mg/kg and in about half the experiments it became necessary to infuse a further 3 mg/kg or more later. The carotid artery was cannulated for blood pressure measurement and to enable samples for blood gas and isotope analysis to be withdrawn from a three-way tap connexion. It was found helpful to stabilize maternal blood pressure during the caesarean section by slowly infusing about 20 ml. citrated blood from a donor guinea-pig. The infusion was via the right jugular vein, which was also utilized to infuse radioisotopes during the ensuing experiments. After surgery and infusion of blood, maternal blood pressure was normally about 80/60 mmHg and these values were maintained throughout the experiment. In twelve experiments the left jugular vein was cannulated with polythene tubing containing saturated KCl solution jelled in 4% agar which was used for measuring the potential differences between maternal blood and fetal perfusion fluid (see below). Perfusion of the umbilical circulation was carried out according to the technique of Reynolds & Young (1971) with the sow lying in a bath of 0.9% saline maintained at 37 'C. Effluent from the umbilical vein was collected over known times as drops into weighed vials for analysis. The composition of the perfusion solution was NaCl, 120 mM; KCl, 5 mm; CaCl2, 1-25 mM; NaHCO03 25 mm; MgCl2, 0 75 mm; glucose, 5-55 mm; dextran of 40,000 mol. wt., 30 g/l. (Rheomacrodex dextran 40, Pharmacia). During perfusion of the umbilical arteries with this fluid at 37 'C, the pH of the umbilical venous effluent was found to be 7-47. During perfusion, inflow pressure was measured by pressure transduction from a side-arm sited just proximal to the PP 10 tubing in the umbilical arteries. This was usually about 75 mmHg at the standard perfusion rate (0in) of 1-4 ml./min from a calibrated Harvard peristaltic pump. The inflow pressure was subject to variation 30 mmHg in either direction, particularly if a cannula became misaligned. The measured pressure drop in the two lengths of PP 10 tubing was 25 mmHg, giving a usual umbilical arterial pressure of about 50 mmHg. The umbilical venous drainage led into wide-bore tubing of internal diameter 1-6 mm, first polythene then softer transmission tubing. Since the open end of the softer tubing was maintained at the level of the bath fluid, i.e. about 1 cm below the maternal heart, the umbilical venous pressure cannot have been greatly above atmospheric. Experimental procedure. The fetal side of the placental circulation was allowed to equilibrate with perfusion fluid for 10-40 min during which time the outflow from the umbilical vein in was determined. After equilibration, 22NaCl or other radioactive tracer was added ml./min to the perfusion fluid to give a concentration of about 0 1 uc/rml. and serial samples of perfusion fluid collected in tared vials for weighing and isotope analysis. Simultaneously with the start of
(ofa)
PLACENTAL CATION TRANSPORT
47
the radioactive perfusion, an i.V. infusion of 24Na or other radioactive tracer was made from a Harvard syringe pump. This was done at an initially fast but rapidly diminishing rate. An infusion schedule had been previously determined by trial and error which maintained a level of the isotope in maternal plasma constant within 10% of the mean (Bradbury & Kleeman, 1967). Transport of 3H2O across the placenta was used as an index of maternal blood-flow (Bailey, 1974). In most experiments, a near constant concentration of 3H20 in maternal blood was obtained by an initial dose of 25 ml. isotonic saline containing 2 4c/ml. infused at 0-5 ml./min during surgery (20-40 min). This was followed by maintenance infusion of 0-25 #sc/min in saline at the same rate for the duration of the experiment, 2 hr. Samples (2 ml.) of maternal blood were withdrawn into a glass syringe at 30 muin intervals for blood gas and isotope analysis. In later experiments, placental transfer of potassium was measured using the same technique but replacing 22Na and 24Na with 42K and 86Rb in the respective perfusion or infusion fluids. In both Na and K experiments, possible isotope effects were checked by reversing the direction of flow of each isotope in some experiments. No significant differences were found between 22Na and 24Na or 42K and "6Rb, in this preparation. Lithium extraction was estimated by replacing 5mM-NaCl in the perfusion fluid with LiCl, and fractional entry in separate experiments by infusing isotonic LiCl I.v. into the mother to give maternal arterial concentration of about 4 mM. The effect of ouabain on Na or K exchange across the placenta was studied by dividing the experiment in two sections, an initial control period of 1 hr in which unidirectional sodium or potassium movement were monitored, followed by a second section of 1 hr in which ouabain was added to the fetal perfusion fluid to give a final concentration of 10-5 or 5 x 10-5 M. In some experiments, the effect of a low sodium perfusion fluid, NaCl component replaced with choline chloride was tested by perfusing this fluid for 40 min either immediately before or after the control fluid. Ouabain 10-5 M in normal fluid was perfused over the final 40 min period. Tritiated water transfer was measured in all situations. After infusing for a total of 2 hr, the experiment was terminated and the placenta was usually removed for isotope analysis. The umbilical and uterine vessels were tied off close to the placenta, which was lifted out on to a Petri dish and gently blotted to remove bath water. Pieces of suitable size for analysis, 1 g for y-emittors, were placed in appropriately labelled tared vials and isotope analysis carried out (see- below). The electrical potential difference between maternal blood and fetal perfusion fluid was measured by means of bridges made from 4% agar jelled in saturated KCl solution. The bridges for maternal jugular vein and fetal perfusion fluid reservoir led each to one calomel-mercury half-cell. A lead from each half-cell connected to input of Vibron high impedance electrometer. I8otope and chemical aralysi. Weighed tissue samples and 1 ml. aliquots of weighed liquid samples containing y-emitting isotopes were counted in a Panax automatic scintillation counter. Separation of 22Na and 24Na activities were made by initially counting in a 'window' set to exclude most of the 22Na emission. After decay of 24Na, the 22Na were counted at maximum efficiency. Use of pure 22Na standards allowed cross-over of 22Na emission into the 24Na 'window' to be subtracted. Emission of fi particles was measured in a three-channel Packard Tricarb scintillation counter. The scintillation fluid was prepared by dissolving 6 g of 2,5-diphenyloxazole and 0-05 g of 1,4di[2-(5-phenyloxazolyl)] benzene in 700 ml. of 'Analar' toluene and 300 ml. Analar 2-ethoxyethanol. Samples of 0-1 ml. perfusion fluid or of plasma were added to 15 ml. of this fluid for counting. For y-counting the placenta was cut into pieces weighing approximately 1 g. These were placed in tared Panax vials. The remaining blood and fluid were washed into further vials with isotonic saline. Sodium, K and Li in plasma and perfusion fluid were estimated with an IL 343 flame photometer.
Mathematical analy8i8 of results. When a radioactive solute was present in the perfusion fluid C,, the radioactive mass,, M, lost from the fluid in unit time, was readily calculated from the relation QC (1) Am = (s-Co) at concentration,
where CO was the concentration in the outflow from the umbilical vein, inflow and outflow were found to be equal under steady conditions, and hence the directly measured outflow volume per unit time gave Q1,,
48
D. J. BAILEY AND OTHERS
Similarly, when radioisotope was administered into the maternal bloodstream, giving an arterial concentration, C., and the inflowing perfusion fluid contained no isotope.
Xf C, *Q.f. =
(2)
The main barrier to transport across the guinea-pig placenta is almost certainly the syncytiotrophoblast, the endothelium of the fetal capillaries providing little hindrance to diffusion. Apparent permeability-surface area products, P's, with units of volume, time-' may be obtained by dividing l0fm and M9f by Ci and Ca respectively.
and
P'rm = [(C1-C0)/C1]f Ifm
CO. Q
(3)
(4)
If may be noted that if perfusion flow is the same in all experiments as here, extractions, (Ci -CO) Cl, will be directly related to Pi s and fractional entry Co/Ca to PS s by the same constant. Also relative P's in one direction will be equal to relative time permeabilities in the same direction provided transfer of solute across the placenta is small relative to the umbilical perfusion flow and the maternal blood flow. Condition of animals and placentae. The partial pressures of 02 and CO2 in arterial blood were generally well maintained during the experiments. At the end of thirty-four experiments, the mean arterial pH was 7-36; the Po2o, 35-2 mmHg and the P02, 79.7 mmHg, these parameters having changed little over the previous 2 hr. The fractional entry CQ/Ca for 3H20 was frequently one or close to one and rarely below 0-60. The occurrence of Co/Ca s of unity and other evidence have suggested that the guinea-pig placenta functions as a counter-current exchange (Bailey, 1974). Values of Co/Ca below 0-5 were considered to represent inadequate placenta perfusion with maternal blood. Perfusion of 1O-5 M-ouabain always resulted in a slow rise in umbilical arterial pressure, usually about 40 mmlHg over the hour. There was sometimes a measurable increase in maternal arterial pressure or a reduction in 3H20 transfer (see Results) or both. During perfusion of 5 x 10-5 Mouabain, these effects always occurred, were more marked and were not infrequently associated with the final stopping of the maternal heart. In the few experiments in which the placenta was perfused with a low sodium fluid, provided that the sodium concentration in the inflowing fluid was dropped gradually over 10 min, there was no consistent change in umbilical perfusion pressure or in maternal blood pressure. There was usually a decrease in 3H20 transfer (see Results). RESULTS
Placental transport of Na and Li. Fractional entry and extraction of Na into and from fetal perfusion fluid was measured simultaneously by the use of 22Na and 24Na tracers. Fig. 1 shows that steady states for both entry and extraction were reached within 10-20 min. Fractional entry had a mean value of 0-109 + 0-009 (s.E. of mean) (n = 11) and extraction 0-119 + 0-013 (n = tO). The values obtained were not influenced by whether 22Na or 24Na was present in the maternal circulation or perfusion fluid. The results show the absence of a significant difference between rates of entry and exit although within 95 % confidence limits a difference of up to 0-030 or 26 % in either direction might remain undetected. Infusion of LiCl and substitution of 5 mM-LiCl in the perfusion fluid allowed estimation of bidirectional permeability to this ion in separate experiments. Mean fractional entry was 0-061 + 0-011 (4), which was 56 % of Na entry. Extraction of lithium was 0-096 + 0-009 (7); the difference between entry and extraction was barely significant (P = 0-05). Perfusion with 10-1 m-ouabain had no effect on fractional entry or exit of 24Na
49 PLACENTAL CATION TRANSPORT (Tables 1 and 2). In five experiments, results not tabulated, a perfusion fluid in which the sodium concentration was reduced to 17'5 mm by choline replacement was
substituted for a normal fluid for 40 min and vice versa. The 24Na extraction was increased from 04102 + 0-010 to 0-131 + 0'014 (P = 0-05-0-02, paired t test). 14
24Na: 1-0
C.
ts
~-_
ts 0
qtwo
06
tf 0-2
24Na: Co/Cc
22Na: (C.-C,)/Ci 40
20
80
60
100
120
Time (min)
Fig. 1. Comparison of C. with C. for 24Na, when a near constant level of this radioisotope was maintained in maternal blood plasma. Extraction,(C, -C.)/C1, of 22Na from umbilical perfusion fluid measured in the same experiment is also included. Values of C. at each time, expressed as ratio to mean of values at all times.
08 u
(5 ~-_ °
046
vol
I
co0
I
Q x
w
--l
%
-
_-0 m
a -
0A2Z
a U
asI-1
--F.
n
Ouabain
-a
1O-sM
I
I
40
6;0 Time (min)
80
100
120
Fig. 2. Extraction (C1 C.) of 42K from umbilical perfusion fluid. At 1 hr a fluid containing ouabain 10-5 M was substituted for the normal one. The results from a similar experiment, completely without ouabain, are included for comparison (interrupted line). -
D. J. BAILEY AND OTHERS Placental transport of potassium and the effect of ouabain. Fractional entry and extraction of potassium was measured using 42K or 86Rb. The placenta appeared to be unable to discriminate between the two isotopes. Fractional entry for 42K and "Rb was consistently greater than the extraction for these two isotopes. Thus the mean fractional entry (combined values, 1st hour) was 0-364 + 0-020(9) (Table 1), whereas the corresponding mean extraction was 0-288 + 0-017 (13), Table 3 (P=0-01-0-002). 50
TABLE 1. Fractional entry of 24Na, 42K and 86Rb, maintained at stable level in maternal blood plasma, into the umbilical perfusion fluid, measured during the first and second hours. Either a normal fluid was perfused throughout or was exchanged for one containing 10-5 M or 5 x 1O-5Mouabain during the second hour. Limits are standard errors. Where mean is from two experiments, individual results are given in parentheses Fractional entry from blood to perfusion fluid (Co/Ca)
perfusion
CO/CE for
CO/C for
cation - 1-0+ 9-6
3H20 -7-6+5-1
-0-9+3-7
- 32-2 + 14-2
+ 8-7 + 7-2 +7-4
- 21-1
24Na
4
0-102+0-013 0-101+ 0 023
42K
3
86Rb
2
0-318 + 0-043 0-361 ± 0-058 0-330 (0-309, 0-336 (0-316,
5
Ouabain 10-5 M during second hour Ouabain 5x 10-6 M during second hour
v8. first hour
Isotope Number First hour Second hour 24Na 7 0-110+0-010 0-109+0-009
r~~~~~~~
Conditions Normal perfusion Ouabain 10-5 M during second hour Normal
% difference, second hour
86Rb
2
86Rb
2
0-356) 0-350) 0-322 + 0-027 0-350 ± 0-035 0-390 (0-402, 0-422 (0-455, 0-377) 0-389) 0-442 (0-443,
0-148 (0-158,
0-441)
0-139)
- 66-4
-4-2+0-5
-44.9
Permeability to K was greater than that to Na and, unlike Na, K exchange was inhibited by ouabain (Fig. 2). K extraction was reduced by 43 % in the presence of 10- m-ouabain (P < 0-001, paired t test, Table 2), and entry was reduced by 66 % in two experiments with 5 x 10-5 M-ouabain in the fetal perfusion fluid (Table 1). At a concentration of 10-5 M, the effect of ouabain on 3H0O exchange was rather variable but generally reduced it somewhat (Tables 1 and 2); at 5 x 10-5 M, ouabain caused a more marked reduction of 45 % in CO/OC for 3H20 in two experiments (Table 1). During perfusion with 5 x 10-5 M-Ouabain in the fetal fluid the maternal blood pressure rose, indicating that ouabain had access to the maternal blood-stream. In three experiments in which the high ouabain concentration was used, the investigation had to be abandoned due to maternal cardiac arrest, presumably due to ouabain intoxication. Following experiments with 42K or 8"Rb to measure potassium transport, the
PLACENTAL CATION TRANSPORT 51 placenta was analysed for each isotope and the activity expressed as a ratio to that in the appropriate fluid source of the isotope. During the experiments, K was accumulated by the placenta, reaching a concentration of 3-34 times that in the umbilical perfusion fluid and no less than 11-19 times that in the maternal blood plasma (Table 3). Since the potassium perfusion fluid was 5 0 mm and the mean of maternal plasma 3-4 mm the exchangeable K in the placenta after 2 hr experiment must have been 3-34 x 5-0 + 11-19 x 3-4 = 55 m-mole. kg-. The total K content in TABLE 2. Extractions of 42K and 86Rb from the umbilical perfusion fluid, measured during the first and second hours. Either a normal fluid was perfused throughout or was exchanged for one containing 10-5 M-ouabain during the second hour. In the experiments with 24Na, the periods were 40 min and included 40 min low Na perfusion (not in Table; see text). Limits are standard errors. When mean is for two experiments, individual results are given in parentheses. % difference, second Extraction (C1-Q00)/C1 hours v8. first hour for cations
Conditions Normal perfusion
Ouabain 10-5 M during second hour
Isotope 42K
86Rb Results for 42K and 86Rb combined 42K 86Rb
Number First hour Second hour 2 0-268 (0-276, 0-248 (0-254, 0-242) 0.260) 4 0-267 + 0-046 0*262 ± 0 035 0-266+ 0033 0-253+ 0-029 6
Results for 42K and 86Rb combined 24Na
(C1-C0/C,
CA/C.
-48 +51
-8-4 +28
for cations
4 3
0-315+ 0-015 0187+ 0-068 0-297+ 0022 0-161 0-061 -
7
0-306+0-016 0-174+0-063
3
0-101 + 0-017 0-107 + 0 024
- 43-3+6-5
+ 8-5 ± 19-0
3H20
-17-5+8-2
- 28-6 + 18-1
perfused placentae was estimated by flame photometer to be 54 0 m-mole. kg-'. The similarity of these values suggests that equilibration of isotopes from both the fetal and maternal side had occurred by 2 hr. Accumulation of 42K and 86Rb into placentae from perfusion fluid was abolished and uptake from maternal blood was very much reduced in the presence of 10-5 M-ouabain in perfusion fluid. When the perfusion fluid was changed from normal to l0-5 M-ouabain, net potassium movement changed from a slight positive extraction to a net entry into the perfusion fluid, the peak K concentration in the outflow occurring at 10-15 min after the start of the ouabain fluid (Fig. 3). In nine experiments, a net flux of 0'28 + 0*06 /mole. min- (out of fluid) was converted to a net flux of 0-71 + 0415 (into perfusion fluid), this estimated during the first 20 min of ouabain perfusion. Potential difference across the placenta. In twelve control experiments the transplacental p.d. was measured soon after the beginning of the experiment and after 2 hr perfusion. There was no tendency for p.d. to change during this experimental period, and the mean value for the twelve experiments was 6.3 + 0 8 mV (fetus
D. J. BAILEY AND OTHERS
52
TABLE 3. Relative radioactivity in perfused placentae after 42K or 86Rb had been perfused through the umbilical circulation (fourth column) or maintained at a stable concentration in blood plasma (sixth column). Either a normal fluid was perfused throughout or was exchanged for one containing ouabain 10-5 or 5 x 10-5 during the second hour. Limits are standard errors. When mean is from two experiments, individual results are given in parentheses.
Conditions Normal
Activity. g-' placenta/activity. mlA.- perfusion Number fluid 4 3*16+0-42 4 3-54 ± 0-74
Isotope 24K 86Rb
perfusion
Ouabain 10-5 M during second hour
9*30±1-79 14-08 (8-36, 19.80) 11-19+2*20
Results for 42K and 86Rb combined
8
3-34+0*43
5
42K
4 3
0-73+0*12
1 1
5*19
0*89±0-12
7
080+0'12
2
6-98
1 1
2*83 5-69
2
4*26
86Rb
Results for 42K and 8"Rb combined 42K
Ouabain 5 X 10-5 M during second hour
No. 3 2
Activity. g-1 placenta/activity. ml.-> plasma
86Rb
Results for 42K and 86Rb combined
8-76
8 7 -
E 0
6
;5
o o ° o
0
0
0
o ° °
1°
,3 4 0 -
3 2
K
Ouabain 10-5M 20
40
60
80
100
120
Time (min)
Fig. 3. K concentration in the perfusion outflow. At 1 hr a perfusion fluid containing 10-5 M-ouabain was substituted for the normal one. Interrupted line is K concentration in inflow.
PLACENTAL CATION TRANSPORT 53 negative). After perfusing with low Na fluid, the mean p.d. in three experiments was 0 3 + 0 4 mV (fetus negative). After 10-5 M-ouabain perfusion for 1 hr the mean p.d. fell to 4-3 + 1 O0mV. DISCUSSION
Estimated unidirectional fluxes Estimation of the exchange of isotope allows calculation of unidirectional fluxes of the ions from mother to fetus, Jmb from the equation Jmf = Ca Pim' where Ca is the total concentration of the ion in arterial plasma of the mother. Similarly the reverse flux, Jfm, is given by Jfm = Ci * P rM, where Ci is the total concentration of the ion in perfusion fluid. These equations neglect back movement of the radioactive ions which in the case of Na will be small. The JMf for Na was calculated to be 22 /tmole. min'. The Na flux in the opposite direction is similar because of the near identity of both the P's and of both the Na ion concentrations. The values for JMf is close to that estimated by Flexner & Pohl (1941), 26 mg. h-1 or 19 /tmole. min-'. They injected 24Na i.v. into maternal guineapigs and removed fetus for counting at set times afterwards. Stulc & Svihovec (1977) found on average Jmf of 13 4amole . min-' and a Jmf of 17 Iamole . min'. Their lower values may be attributed to the pregnancies being at an average earlier stage, 40 days to term, and to their average fetal weights being correspondingly less, 50.2 g as against 69-5 g. The difference between influx and efflux recorded by tulc & gvihovec (1977) cannot, as they point out, occur under normal conditions in the intact animal. The discrepancy between the present results and those of htulc & 9vihovec with respect to identity or non-identity of influx and efflux may be associated with the magnitude of the transplacental potential difference measured in each case, see below. All these values are some 2-4 times larger than the Na fluxes measured in the guinea-pig placenta perfused in vitro through both maternal and fetal circulation (Schrdder et al. 1972). Unidirectional K fluxes in our preparation were estimated at 1-7 and 1-8 molel. mind from mother to fetus and in the opposite direction respectively. The higher P' from mother to fetus (Table 1) is balanced by a lower K concentration (3.4 mM) in maternal arterial plasma than in the perfusion fluid (5 mM). The end-result is similar unidirectional fluxes. The magnitude of these is almost certainly underestimated because capillary concentrations of the radioisotopes will at the rates of exchange be significantly different from either C. or C,. The authors know of no other estimates of transplacental K fluxes in the literature.
Transplacental p.d. and relative permeabilities Mellor (1969) recorded a transplacental electrical potential of - 18 mV in the intact anaesthetized guinea-pig. Similar values have been obtained from the guinea-pig placenta perfused in situ when the exposed uterus and posterior part of the mother was immersed in liquid paraffin at constant temperature rather than in isotonic saline (9tulc & 9vihovec, 1973, 1977). It seems likely that the smaller values of the
D. J. BAILEY AND OTHERS p.d. recorded in the present experiments were due to short-circuiting in relation to the saline. Although there is an apparent reduction in the absolute size of the potential, the present results are comparable to those of 8tulc & 8vihovec (1973) in showing a big shift towards the positive due to a low sodium fluid and no great change due to ouabain. If passive independent transport is assumed, apparent permeability - surface area products (P) as defined in the Methods, may be multiplied by VF/RT/ exp(VR/RT) -1 to correct for the influence of the transepithelial potential difference (V) on monovalent cation movement (Schultz & Zalusky, 1964). This has 54
TABLE 4. Relative permeability of placenta to cations in both directions. Mean values for PS-products corrected for transplacental potential difference and expressed relative to Na entry as unity. Relative permeabilities
Cation Li Na K-Rb
Mother to fetus 0-55
Fetus to mother 0-97
1-00
1120
3-20
2-55
been done for mean values of P in both directions in association with a mean transplacental p.d. of 6-3 mV (fetus negative) and the result expressed relative to the corrected PS-product for radioactive Na from mother to fetus as unity. Measures of relative permeabilities in both directions are thus obtained, Table 4. The placenta exhibits considerable selectivity with respect to transport of the four cations studied. For transport both towards and away from the fetus, the selectivity is Rb K > Na > Li. Whilst the sequences are the same for each direction, the sizes of the ratios differ more markedly for transport from mother to fetus. The ratios suggest net active movement of Na from mother to fetus, and of net active transport of Li and K in the reverse direction. Such a distribution of active transport is compatible with the pressure of a dominant conventional Na-K pump in the placenta, directed with its active Na component towards the mother (Atulc & Avihovec, 1977). -
Significance of ouabain effects The question may be asked as to whether the changes in K and Rb movement associated with ouabain in the perfusion fluid are due to specific inhibition of transport, since even at the lower concentration of 1O-5 M this compound has some influence on the placental circulations. The slow rise in perfusion pressure indicates a proportionate increase in vascular resistance on the umbilical side, but it developed more slowly than the effect on 42K and "Rb extraction and on net K movement. Whilst fractional entry of 3H20 was generally somewhat reduced by 10-5 M-ouabain, the effect was variable and in the experiments on 42K and 86Rb extraction very small compared with that on the cation extractions. The time course of the umbilical vascular effect and the small relative size of the maternal effect, taken together with the striking absence of any reduction in sodium movement in either direction, due to 10-5 m-ouabain strengthen the belief that the inhibitor is also acting specifically on
PLACENTAL CATION TRANSPORT 55 potassium and rubidium transport. At 5 x 10-5 M, ouabain caused a profound decrease in maternal blood flow, but it is perhaps significant that fractional entry of 86Rb was more markedly reduced. Model for transplacental transport in guinea-pig Experiments on the transport of polar non-electrolytes of differing molecular weight across the guinea-pig placenta in both the intact and the perfused in situ conditions (Faber, 1973; M. W. B. Bradbury, V. M. France & R. Hedley, unpublished observations) indicate a water-filled transplacental route. There is a direct relation between transplacental permeability and free diffusion coefficient in water. Such observations imply the presence of effective water-filled channels of large diameter, 10 nm or more. A significant fraction of cation transport may follow this pathway, but the degree of cation selectivity proves that a more specific route must also exist. A high proportion of the transplacental fluxes of 42K and 86Rb might be transcellular. The active components of these fluxes would involve some ouabain-dependent potassium pumping at the fetal-facing membrane of the syncytiotrophoblast. The evidence for such a pump at this membrane is inhibition by ouabain of 42K and 86Rb extraction from the perfusion fluid; blocking of 42K and 86Rb accumulation from the perfusion fluid by ouabain; and induction by ouabain of a net flux of K into the perfusion fluid. The less definitive evidence for a K pump directed to the opposite sense at the maternal-facing membrane of the trophoblast is greater accumulation of 42K and 86Rb by the placenta from its maternal rather than its fetal side, greater reduction of fractional entry of 86Rb than of 3H20 by 5 x 10-5mi-ouabain and histochemical demonstration of maximum density of Na-K-adenosine triphosphatase in relation to the maternal microvilli (Firth & Farr, 1977). The negative potential may then be tentatively attributed, as was done by gtulc & Svihovec (1973), to dominant electrogenic pumping of sodium ions at the maternal facing membrane of the syncytiotrophoblast. Since the low Na fluid must deplete intracellular Na, the active electrogenicity will be reduced. Since ouabain in the perfusion fluid must primarily affect the pump in the fetal facing membrane, the potential will be unchanged or even increased. Although the presence of fixed waterfilled channels might have escaped detection in electron micrographs, such low resistance pathways for current flow would hardly support a transplacental potential difference of the size measured. Opening and closing vesicles or tubules would have a higher resistance. This work was supported by an M.R.C. project grant. REFERENCES
BAILEY, D. J. (1974). Counter current flow of maternal and foetal bloodstreams in the guineapig placenta. J. Physiol. 242, 104- 105P. BAWDEN, J. W. & WYOLKOFF, A. S. (1967). Foetal blood calcium response to maternal calcium infusion in sheep. Am. J. Obstet. Gynec. 99, 55-60. BRADBURY, M. WV. B., CROWDER, J., DESAI, S., REYNOLDS, J. M., REYNOLDS, M. & SAUNDERS, N. R. (1972). Electrolytes and water in the brain and cerebrospinal fluid of the foetal sheep and guinea-pig. J. Physiol. 227, 591-610.
D. J. BAILEY AND OTHERS
56
BRADBURY, M. W. B., FRANCE, V. M., HEDLEY, R. & PARRY, H. (1977). Transport of cations by the guinea-pig placenta perfused in situ. J. Physiol. 272, 22-23P. BRADBURY, M. W. B. & KLEEMAN, C. R. (1967). Stability of the potassium content of cerebrospinal fluid and brain. Am. J. Physiol. 213, 519-528. FABER, J. J. (1973). Diffusional exchange between foetus and mother as a function of the physical properties of the diffusing molecules. In Sir Joseph Barcroft Centenary Symposium on Foetal and Neonatal Physiology, ed. COMLINE, R. S., CROSS, K. W., DAWES, G. S. & NATHANIELSZ, P. W., pp. 306-327. Cambridge: Cambridge University Press. FmRTH, J. A. & FARR, A. (1977). Structural features and quantitative age-dependent changes in the intervascular barrier of the guinea-pig haemochorical placenta. Cell Tissue Res. 184, 507-516. FLEXNER, L. B. & POHL, H. A. (1941). Transfer of radioactive sodium across the placenta of the guinea-pig. Am. J. Physiol. 132, 594-606. GREESON, D. D., CRAWFORD, E. G., CHANDLER, D. C. & BAWDEN, J. W. (1968). Foetal blood calcium response to maternal hypercalcaemia in guinea-pigs. J. dent. Res. 47, 447-449. MELLOR, D. J. (1969). Potential differences between mother and foetal at different gestational
ages in the rat, rabbit and guinea-pig. J. Physiol. 204, 395-405. MESCHA, G., WOLKOFF, A. S. & BARRON, D. H. (1958). Differences in electrical potential across the placenta of goats. Proc. natn. Acad. Sci. U.S.A. 44, 483-485. MONEY, W. L. & DANCIS, J. (1960). Technique for the in situ study of placental transport in the pregnant guinea-pig. Am. J. Obstet. Gynec. 80, 209-214. REYNOLDS, M. L. & YoUNG, M. (1971). The transfer of free-amino nitrogen across the placental membrane in the guinea-pig. J. Physiol. 214, 583-597. SCHRODER, H., STOLP, W. & LEICHTWEISS, H. P. (1972). Measurements of Na+ transport in the isolated artificially perfused guinea-pig placenta. Am. J. Obstet, Gynec. 114, 51-57. SCHULTZ, S. G. & ZALUSKY, R. (1964). Ion transport in isolated rabbit ileum I. Short circuit current and Na fluxes. J. gen. Physiol. 47, 567-584. SERRANO, C. V., TALBERT, L. M. & WELT, L. G. (1964). Potassium deficiency, in the pregnant dog. J. clin. Invest. 43, 27-31. STEWART, E. L. & WELT, L. G. (1961). Protection of the fetus in experimental potassium
depletion. Am. J. Physiol. 200, 824-826. & gVIHOVEC, J. (1973). Effect of potassium cyanide, strophanthin or sodium free perfusion fluid on the electrical potential difference across the guinea-pig placenta perfused in situ. J. Physiol. 231, 403-415. 9TULC, J. & 9VIHOVEC, J. (1977). Placental transport of sodium in the guinea-pig. J. Physiol. 265, 691-703. WIDDAS, W. F. (1961). Transport mechanisms in the foetus. Br. med. Bull. 17, 107-111.
9TULC, J.
45
CATION TRANSPORT ACROSS THE GUINEA-PIG PLACENTA PERFUSED IN SITU
BY D. J. BAILEY, M. W. B. BRADBURY, VENETIA M. FRANCE, RUTH HEDLEY, SAROJ NAIK AND HELEN PARRY From the Department of Physiology, King's College, Strand, London WC2R 2LS
(Received 30 January 1978) SUMMARY
1. The guinea-pig placenta perfused in situ via the umbilical circulation has been used to measure unidirectional fluxes of Na from mother to fetus, and in the reverse direction, with 24Na and 22Na. There was no significant difference between the two fluxes, each being 22 /tmole. min-. 2. Ouabain 10-5 M in the perfusion fluid had no detectable effect on radioisotopic movement of Na in either direction. 3. Unidirectional fluxes of 42K in both directions were approximately equal at 1*7 ,umole.min-1 mother to fetus and 18 jumole.min-1 in the reverse direction, despite a K concentration of 3-4 mm on the maternal side and 5 0 mm on the fetal side of the placenta. 4. Extraction of 42K and 86Rb from the perfusion fluid was inhibited by 43 % by 10-5 M-ouabain in the fluid. This effect was largely due to a reduction of isotope uptake by the placental tissue. 5. The relative permeabilities of the placenta, mother to fetus, were Rb K (3.2) > Na (1.0) > Li (0.55). 6. Under the experimental conditions, the electrical potential difference between perfusion fluid and maternal blood was 6 mV (fetus negative). It was shifted towards the positive by a low Na fluid. 7. The results suggest the presence of a dominant Na-K pump (active component towards mother) sited at the maternal-facing membrane of the syncytiotrophoblast together with a subsidiary pump oriented in the opposite direction and probably sited at the fetal-facing membrane of the syncytiotrophoblast. 8. A high proportion of Na movement particularly towards the fetus is probably passive, occurring through water-filled spaces, whilst K movement is more dependent on active transport. INTRODUCTION
Mechanisms controlling fetal nutrition and homoeostasis are at present little understood. However, the placenta is generally regarded as being the organ responsible for such control. Early studies demonstrated the presence of a transplacental potential difference (p.d.) (Meschia, Wolkoff & Barron, 1958; Widdas, 1961; Mellor, 1969; of fetal K homoeostasis (Stewart & Welt, 1961; Serrano, Talbert & Welt, 1964)
D. J. BAILEY AND OTHERS and Ca homoeostasis (Bawden & Wolkoff, 1967; Greeson, Crawford, Chandler & Bawden, 1968; Bradbury, Crowder, Desai, Reynolds, Reynolds & Saunders, 1972). Placental Na transport has been studied more recently in the guinea-pig (8tulc & 8vihovec, 1973; 1977), indicating some evidence of active transport from fetus to mother, although earlier studies (Schr6der, Stolp & Leichtweiss, 1972) in the same preparation failed to find conclusive evidence for an active process. The present studies were undertaken to obtain basic facts concerning the transport of four monovalent cations across the guinea-pig placenta. The results have allowed construction of a model consistent with our present understanding of electrolyte exchange. Such a model is an essential preliminary to full characterization of the homoeostatic mechanism for ions. A preliminary account of this work has been given before the Physiological Society (Bradbury, France, Hedley & Parry, 1977). 46
METHODS
Preparation of the pregnant guinea-pig. Pregnant guinea-pigs at 59-61 days' gestation were delivered by British Rail from Redfern Animal Breeders Ltd., Brenchley, Kent, from 1 to 5 days before the experiment. The weighed guinea-pig was gently restrained while lignocaine (Lidothane) was injected s.c. into the lower forelimb. A dilute solution of pentobarbitone (10 mg/ml.) was slowly infused into the exposed brachial vein over a period of 15 min. The normal dose was 25 mg/kg and in about half the experiments it became necessary to infuse a further 3 mg/kg or more later. The carotid artery was cannulated for blood pressure measurement and to enable samples for blood gas and isotope analysis to be withdrawn from a three-way tap connexion. It was found helpful to stabilize maternal blood pressure during the caesarean section by slowly infusing about 20 ml. citrated blood from a donor guinea-pig. The infusion was via the right jugular vein, which was also utilized to infuse radioisotopes during the ensuing experiments. After surgery and infusion of blood, maternal blood pressure was normally about 80/60 mmHg and these values were maintained throughout the experiment. In twelve experiments the left jugular vein was cannulated with polythene tubing containing saturated KCl solution jelled in 4% agar which was used for measuring the potential differences between maternal blood and fetal perfusion fluid (see below). Perfusion of the umbilical circulation was carried out according to the technique of Reynolds & Young (1971) with the sow lying in a bath of 0.9% saline maintained at 37 'C. Effluent from the umbilical vein was collected over known times as drops into weighed vials for analysis. The composition of the perfusion solution was NaCl, 120 mM; KCl, 5 mm; CaCl2, 1-25 mM; NaHCO03 25 mm; MgCl2, 0 75 mm; glucose, 5-55 mm; dextran of 40,000 mol. wt., 30 g/l. (Rheomacrodex dextran 40, Pharmacia). During perfusion of the umbilical arteries with this fluid at 37 'C, the pH of the umbilical venous effluent was found to be 7-47. During perfusion, inflow pressure was measured by pressure transduction from a side-arm sited just proximal to the PP 10 tubing in the umbilical arteries. This was usually about 75 mmHg at the standard perfusion rate (0in) of 1-4 ml./min from a calibrated Harvard peristaltic pump. The inflow pressure was subject to variation 30 mmHg in either direction, particularly if a cannula became misaligned. The measured pressure drop in the two lengths of PP 10 tubing was 25 mmHg, giving a usual umbilical arterial pressure of about 50 mmHg. The umbilical venous drainage led into wide-bore tubing of internal diameter 1-6 mm, first polythene then softer transmission tubing. Since the open end of the softer tubing was maintained at the level of the bath fluid, i.e. about 1 cm below the maternal heart, the umbilical venous pressure cannot have been greatly above atmospheric. Experimental procedure. The fetal side of the placental circulation was allowed to equilibrate with perfusion fluid for 10-40 min during which time the outflow from the umbilical vein in was determined. After equilibration, 22NaCl or other radioactive tracer was added ml./min to the perfusion fluid to give a concentration of about 0 1 uc/rml. and serial samples of perfusion fluid collected in tared vials for weighing and isotope analysis. Simultaneously with the start of
(ofa)
PLACENTAL CATION TRANSPORT
47
the radioactive perfusion, an i.V. infusion of 24Na or other radioactive tracer was made from a Harvard syringe pump. This was done at an initially fast but rapidly diminishing rate. An infusion schedule had been previously determined by trial and error which maintained a level of the isotope in maternal plasma constant within 10% of the mean (Bradbury & Kleeman, 1967). Transport of 3H2O across the placenta was used as an index of maternal blood-flow (Bailey, 1974). In most experiments, a near constant concentration of 3H20 in maternal blood was obtained by an initial dose of 25 ml. isotonic saline containing 2 4c/ml. infused at 0-5 ml./min during surgery (20-40 min). This was followed by maintenance infusion of 0-25 #sc/min in saline at the same rate for the duration of the experiment, 2 hr. Samples (2 ml.) of maternal blood were withdrawn into a glass syringe at 30 muin intervals for blood gas and isotope analysis. In later experiments, placental transfer of potassium was measured using the same technique but replacing 22Na and 24Na with 42K and 86Rb in the respective perfusion or infusion fluids. In both Na and K experiments, possible isotope effects were checked by reversing the direction of flow of each isotope in some experiments. No significant differences were found between 22Na and 24Na or 42K and "6Rb, in this preparation. Lithium extraction was estimated by replacing 5mM-NaCl in the perfusion fluid with LiCl, and fractional entry in separate experiments by infusing isotonic LiCl I.v. into the mother to give maternal arterial concentration of about 4 mM. The effect of ouabain on Na or K exchange across the placenta was studied by dividing the experiment in two sections, an initial control period of 1 hr in which unidirectional sodium or potassium movement were monitored, followed by a second section of 1 hr in which ouabain was added to the fetal perfusion fluid to give a final concentration of 10-5 or 5 x 10-5 M. In some experiments, the effect of a low sodium perfusion fluid, NaCl component replaced with choline chloride was tested by perfusing this fluid for 40 min either immediately before or after the control fluid. Ouabain 10-5 M in normal fluid was perfused over the final 40 min period. Tritiated water transfer was measured in all situations. After infusing for a total of 2 hr, the experiment was terminated and the placenta was usually removed for isotope analysis. The umbilical and uterine vessels were tied off close to the placenta, which was lifted out on to a Petri dish and gently blotted to remove bath water. Pieces of suitable size for analysis, 1 g for y-emittors, were placed in appropriately labelled tared vials and isotope analysis carried out (see- below). The electrical potential difference between maternal blood and fetal perfusion fluid was measured by means of bridges made from 4% agar jelled in saturated KCl solution. The bridges for maternal jugular vein and fetal perfusion fluid reservoir led each to one calomel-mercury half-cell. A lead from each half-cell connected to input of Vibron high impedance electrometer. I8otope and chemical aralysi. Weighed tissue samples and 1 ml. aliquots of weighed liquid samples containing y-emitting isotopes were counted in a Panax automatic scintillation counter. Separation of 22Na and 24Na activities were made by initially counting in a 'window' set to exclude most of the 22Na emission. After decay of 24Na, the 22Na were counted at maximum efficiency. Use of pure 22Na standards allowed cross-over of 22Na emission into the 24Na 'window' to be subtracted. Emission of fi particles was measured in a three-channel Packard Tricarb scintillation counter. The scintillation fluid was prepared by dissolving 6 g of 2,5-diphenyloxazole and 0-05 g of 1,4di[2-(5-phenyloxazolyl)] benzene in 700 ml. of 'Analar' toluene and 300 ml. Analar 2-ethoxyethanol. Samples of 0-1 ml. perfusion fluid or of plasma were added to 15 ml. of this fluid for counting. For y-counting the placenta was cut into pieces weighing approximately 1 g. These were placed in tared Panax vials. The remaining blood and fluid were washed into further vials with isotonic saline. Sodium, K and Li in plasma and perfusion fluid were estimated with an IL 343 flame photometer.
Mathematical analy8i8 of results. When a radioactive solute was present in the perfusion fluid C,, the radioactive mass,, M, lost from the fluid in unit time, was readily calculated from the relation QC (1) Am = (s-Co) at concentration,
where CO was the concentration in the outflow from the umbilical vein, inflow and outflow were found to be equal under steady conditions, and hence the directly measured outflow volume per unit time gave Q1,,
48
D. J. BAILEY AND OTHERS
Similarly, when radioisotope was administered into the maternal bloodstream, giving an arterial concentration, C., and the inflowing perfusion fluid contained no isotope.
Xf C, *Q.f. =
(2)
The main barrier to transport across the guinea-pig placenta is almost certainly the syncytiotrophoblast, the endothelium of the fetal capillaries providing little hindrance to diffusion. Apparent permeability-surface area products, P's, with units of volume, time-' may be obtained by dividing l0fm and M9f by Ci and Ca respectively.
and
P'rm = [(C1-C0)/C1]f Ifm
CO. Q
(3)
(4)
If may be noted that if perfusion flow is the same in all experiments as here, extractions, (Ci -CO) Cl, will be directly related to Pi s and fractional entry Co/Ca to PS s by the same constant. Also relative P's in one direction will be equal to relative time permeabilities in the same direction provided transfer of solute across the placenta is small relative to the umbilical perfusion flow and the maternal blood flow. Condition of animals and placentae. The partial pressures of 02 and CO2 in arterial blood were generally well maintained during the experiments. At the end of thirty-four experiments, the mean arterial pH was 7-36; the Po2o, 35-2 mmHg and the P02, 79.7 mmHg, these parameters having changed little over the previous 2 hr. The fractional entry CQ/Ca for 3H20 was frequently one or close to one and rarely below 0-60. The occurrence of Co/Ca s of unity and other evidence have suggested that the guinea-pig placenta functions as a counter-current exchange (Bailey, 1974). Values of Co/Ca below 0-5 were considered to represent inadequate placenta perfusion with maternal blood. Perfusion of 1O-5 M-ouabain always resulted in a slow rise in umbilical arterial pressure, usually about 40 mmlHg over the hour. There was sometimes a measurable increase in maternal arterial pressure or a reduction in 3H20 transfer (see Results) or both. During perfusion of 5 x 10-5 Mouabain, these effects always occurred, were more marked and were not infrequently associated with the final stopping of the maternal heart. In the few experiments in which the placenta was perfused with a low sodium fluid, provided that the sodium concentration in the inflowing fluid was dropped gradually over 10 min, there was no consistent change in umbilical perfusion pressure or in maternal blood pressure. There was usually a decrease in 3H20 transfer (see Results). RESULTS
Placental transport of Na and Li. Fractional entry and extraction of Na into and from fetal perfusion fluid was measured simultaneously by the use of 22Na and 24Na tracers. Fig. 1 shows that steady states for both entry and extraction were reached within 10-20 min. Fractional entry had a mean value of 0-109 + 0-009 (s.E. of mean) (n = 11) and extraction 0-119 + 0-013 (n = tO). The values obtained were not influenced by whether 22Na or 24Na was present in the maternal circulation or perfusion fluid. The results show the absence of a significant difference between rates of entry and exit although within 95 % confidence limits a difference of up to 0-030 or 26 % in either direction might remain undetected. Infusion of LiCl and substitution of 5 mM-LiCl in the perfusion fluid allowed estimation of bidirectional permeability to this ion in separate experiments. Mean fractional entry was 0-061 + 0-011 (4), which was 56 % of Na entry. Extraction of lithium was 0-096 + 0-009 (7); the difference between entry and extraction was barely significant (P = 0-05). Perfusion with 10-1 m-ouabain had no effect on fractional entry or exit of 24Na
49 PLACENTAL CATION TRANSPORT (Tables 1 and 2). In five experiments, results not tabulated, a perfusion fluid in which the sodium concentration was reduced to 17'5 mm by choline replacement was
substituted for a normal fluid for 40 min and vice versa. The 24Na extraction was increased from 04102 + 0-010 to 0-131 + 0'014 (P = 0-05-0-02, paired t test). 14
24Na: 1-0
C.
ts
~-_
ts 0
qtwo
06
tf 0-2
24Na: Co/Cc
22Na: (C.-C,)/Ci 40
20
80
60
100
120
Time (min)
Fig. 1. Comparison of C. with C. for 24Na, when a near constant level of this radioisotope was maintained in maternal blood plasma. Extraction,(C, -C.)/C1, of 22Na from umbilical perfusion fluid measured in the same experiment is also included. Values of C. at each time, expressed as ratio to mean of values at all times.
08 u
(5 ~-_ °
046
vol
I
co0
I
Q x
w
--l
%
-
_-0 m
a -
0A2Z
a U
asI-1
--F.
n
Ouabain
-a
1O-sM
I
I
40
6;0 Time (min)
80
100
120
Fig. 2. Extraction (C1 C.) of 42K from umbilical perfusion fluid. At 1 hr a fluid containing ouabain 10-5 M was substituted for the normal one. The results from a similar experiment, completely without ouabain, are included for comparison (interrupted line). -
D. J. BAILEY AND OTHERS Placental transport of potassium and the effect of ouabain. Fractional entry and extraction of potassium was measured using 42K or 86Rb. The placenta appeared to be unable to discriminate between the two isotopes. Fractional entry for 42K and "Rb was consistently greater than the extraction for these two isotopes. Thus the mean fractional entry (combined values, 1st hour) was 0-364 + 0-020(9) (Table 1), whereas the corresponding mean extraction was 0-288 + 0-017 (13), Table 3 (P=0-01-0-002). 50
TABLE 1. Fractional entry of 24Na, 42K and 86Rb, maintained at stable level in maternal blood plasma, into the umbilical perfusion fluid, measured during the first and second hours. Either a normal fluid was perfused throughout or was exchanged for one containing 10-5 M or 5 x 1O-5Mouabain during the second hour. Limits are standard errors. Where mean is from two experiments, individual results are given in parentheses Fractional entry from blood to perfusion fluid (Co/Ca)
perfusion
CO/CE for
CO/C for
cation - 1-0+ 9-6
3H20 -7-6+5-1
-0-9+3-7
- 32-2 + 14-2
+ 8-7 + 7-2 +7-4
- 21-1
24Na
4
0-102+0-013 0-101+ 0 023
42K
3
86Rb
2
0-318 + 0-043 0-361 ± 0-058 0-330 (0-309, 0-336 (0-316,
5
Ouabain 10-5 M during second hour Ouabain 5x 10-6 M during second hour
v8. first hour
Isotope Number First hour Second hour 24Na 7 0-110+0-010 0-109+0-009
r~~~~~~~
Conditions Normal perfusion Ouabain 10-5 M during second hour Normal
% difference, second hour
86Rb
2
86Rb
2
0-356) 0-350) 0-322 + 0-027 0-350 ± 0-035 0-390 (0-402, 0-422 (0-455, 0-377) 0-389) 0-442 (0-443,
0-148 (0-158,
0-441)
0-139)
- 66-4
-4-2+0-5
-44.9
Permeability to K was greater than that to Na and, unlike Na, K exchange was inhibited by ouabain (Fig. 2). K extraction was reduced by 43 % in the presence of 10- m-ouabain (P < 0-001, paired t test, Table 2), and entry was reduced by 66 % in two experiments with 5 x 10-5 M-ouabain in the fetal perfusion fluid (Table 1). At a concentration of 10-5 M, the effect of ouabain on 3H0O exchange was rather variable but generally reduced it somewhat (Tables 1 and 2); at 5 x 10-5 M, ouabain caused a more marked reduction of 45 % in CO/OC for 3H20 in two experiments (Table 1). During perfusion with 5 x 10-5 M-Ouabain in the fetal fluid the maternal blood pressure rose, indicating that ouabain had access to the maternal blood-stream. In three experiments in which the high ouabain concentration was used, the investigation had to be abandoned due to maternal cardiac arrest, presumably due to ouabain intoxication. Following experiments with 42K or 8"Rb to measure potassium transport, the
PLACENTAL CATION TRANSPORT 51 placenta was analysed for each isotope and the activity expressed as a ratio to that in the appropriate fluid source of the isotope. During the experiments, K was accumulated by the placenta, reaching a concentration of 3-34 times that in the umbilical perfusion fluid and no less than 11-19 times that in the maternal blood plasma (Table 3). Since the potassium perfusion fluid was 5 0 mm and the mean of maternal plasma 3-4 mm the exchangeable K in the placenta after 2 hr experiment must have been 3-34 x 5-0 + 11-19 x 3-4 = 55 m-mole. kg-. The total K content in TABLE 2. Extractions of 42K and 86Rb from the umbilical perfusion fluid, measured during the first and second hours. Either a normal fluid was perfused throughout or was exchanged for one containing 10-5 M-ouabain during the second hour. In the experiments with 24Na, the periods were 40 min and included 40 min low Na perfusion (not in Table; see text). Limits are standard errors. When mean is for two experiments, individual results are given in parentheses. % difference, second Extraction (C1-Q00)/C1 hours v8. first hour for cations
Conditions Normal perfusion
Ouabain 10-5 M during second hour
Isotope 42K
86Rb Results for 42K and 86Rb combined 42K 86Rb
Number First hour Second hour 2 0-268 (0-276, 0-248 (0-254, 0-242) 0.260) 4 0-267 + 0-046 0*262 ± 0 035 0-266+ 0033 0-253+ 0-029 6
Results for 42K and 86Rb combined 24Na
(C1-C0/C,
CA/C.
-48 +51
-8-4 +28
for cations
4 3
0-315+ 0-015 0187+ 0-068 0-297+ 0022 0-161 0-061 -
7
0-306+0-016 0-174+0-063
3
0-101 + 0-017 0-107 + 0 024
- 43-3+6-5
+ 8-5 ± 19-0
3H20
-17-5+8-2
- 28-6 + 18-1
perfused placentae was estimated by flame photometer to be 54 0 m-mole. kg-'. The similarity of these values suggests that equilibration of isotopes from both the fetal and maternal side had occurred by 2 hr. Accumulation of 42K and 86Rb into placentae from perfusion fluid was abolished and uptake from maternal blood was very much reduced in the presence of 10-5 M-ouabain in perfusion fluid. When the perfusion fluid was changed from normal to l0-5 M-ouabain, net potassium movement changed from a slight positive extraction to a net entry into the perfusion fluid, the peak K concentration in the outflow occurring at 10-15 min after the start of the ouabain fluid (Fig. 3). In nine experiments, a net flux of 0'28 + 0*06 /mole. min- (out of fluid) was converted to a net flux of 0-71 + 0415 (into perfusion fluid), this estimated during the first 20 min of ouabain perfusion. Potential difference across the placenta. In twelve control experiments the transplacental p.d. was measured soon after the beginning of the experiment and after 2 hr perfusion. There was no tendency for p.d. to change during this experimental period, and the mean value for the twelve experiments was 6.3 + 0 8 mV (fetus
D. J. BAILEY AND OTHERS
52
TABLE 3. Relative radioactivity in perfused placentae after 42K or 86Rb had been perfused through the umbilical circulation (fourth column) or maintained at a stable concentration in blood plasma (sixth column). Either a normal fluid was perfused throughout or was exchanged for one containing ouabain 10-5 or 5 x 10-5 during the second hour. Limits are standard errors. When mean is from two experiments, individual results are given in parentheses.
Conditions Normal
Activity. g-' placenta/activity. mlA.- perfusion Number fluid 4 3*16+0-42 4 3-54 ± 0-74
Isotope 24K 86Rb
perfusion
Ouabain 10-5 M during second hour
9*30±1-79 14-08 (8-36, 19.80) 11-19+2*20
Results for 42K and 86Rb combined
8
3-34+0*43
5
42K
4 3
0-73+0*12
1 1
5*19
0*89±0-12
7
080+0'12
2
6-98
1 1
2*83 5-69
2
4*26
86Rb
Results for 42K and 8"Rb combined 42K
Ouabain 5 X 10-5 M during second hour
No. 3 2
Activity. g-1 placenta/activity. ml.-> plasma
86Rb
Results for 42K and 86Rb combined
8-76
8 7 -
E 0
6
;5
o o ° o
0
0
0
o ° °
1°
,3 4 0 -
3 2
K
Ouabain 10-5M 20
40
60
80
100
120
Time (min)
Fig. 3. K concentration in the perfusion outflow. At 1 hr a perfusion fluid containing 10-5 M-ouabain was substituted for the normal one. Interrupted line is K concentration in inflow.
PLACENTAL CATION TRANSPORT 53 negative). After perfusing with low Na fluid, the mean p.d. in three experiments was 0 3 + 0 4 mV (fetus negative). After 10-5 M-ouabain perfusion for 1 hr the mean p.d. fell to 4-3 + 1 O0mV. DISCUSSION
Estimated unidirectional fluxes Estimation of the exchange of isotope allows calculation of unidirectional fluxes of the ions from mother to fetus, Jmb from the equation Jmf = Ca Pim' where Ca is the total concentration of the ion in arterial plasma of the mother. Similarly the reverse flux, Jfm, is given by Jfm = Ci * P rM, where Ci is the total concentration of the ion in perfusion fluid. These equations neglect back movement of the radioactive ions which in the case of Na will be small. The JMf for Na was calculated to be 22 /tmole. min'. The Na flux in the opposite direction is similar because of the near identity of both the P's and of both the Na ion concentrations. The values for JMf is close to that estimated by Flexner & Pohl (1941), 26 mg. h-1 or 19 /tmole. min-'. They injected 24Na i.v. into maternal guineapigs and removed fetus for counting at set times afterwards. Stulc & Svihovec (1977) found on average Jmf of 13 4amole . min-' and a Jmf of 17 Iamole . min'. Their lower values may be attributed to the pregnancies being at an average earlier stage, 40 days to term, and to their average fetal weights being correspondingly less, 50.2 g as against 69-5 g. The difference between influx and efflux recorded by tulc & gvihovec (1977) cannot, as they point out, occur under normal conditions in the intact animal. The discrepancy between the present results and those of htulc & 9vihovec with respect to identity or non-identity of influx and efflux may be associated with the magnitude of the transplacental potential difference measured in each case, see below. All these values are some 2-4 times larger than the Na fluxes measured in the guinea-pig placenta perfused in vitro through both maternal and fetal circulation (Schrdder et al. 1972). Unidirectional K fluxes in our preparation were estimated at 1-7 and 1-8 molel. mind from mother to fetus and in the opposite direction respectively. The higher P' from mother to fetus (Table 1) is balanced by a lower K concentration (3.4 mM) in maternal arterial plasma than in the perfusion fluid (5 mM). The end-result is similar unidirectional fluxes. The magnitude of these is almost certainly underestimated because capillary concentrations of the radioisotopes will at the rates of exchange be significantly different from either C. or C,. The authors know of no other estimates of transplacental K fluxes in the literature.
Transplacental p.d. and relative permeabilities Mellor (1969) recorded a transplacental electrical potential of - 18 mV in the intact anaesthetized guinea-pig. Similar values have been obtained from the guinea-pig placenta perfused in situ when the exposed uterus and posterior part of the mother was immersed in liquid paraffin at constant temperature rather than in isotonic saline (9tulc & 9vihovec, 1973, 1977). It seems likely that the smaller values of the
D. J. BAILEY AND OTHERS p.d. recorded in the present experiments were due to short-circuiting in relation to the saline. Although there is an apparent reduction in the absolute size of the potential, the present results are comparable to those of 8tulc & 8vihovec (1973) in showing a big shift towards the positive due to a low sodium fluid and no great change due to ouabain. If passive independent transport is assumed, apparent permeability - surface area products (P) as defined in the Methods, may be multiplied by VF/RT/ exp(VR/RT) -1 to correct for the influence of the transepithelial potential difference (V) on monovalent cation movement (Schultz & Zalusky, 1964). This has 54
TABLE 4. Relative permeability of placenta to cations in both directions. Mean values for PS-products corrected for transplacental potential difference and expressed relative to Na entry as unity. Relative permeabilities
Cation Li Na K-Rb
Mother to fetus 0-55
Fetus to mother 0-97
1-00
1120
3-20
2-55
been done for mean values of P in both directions in association with a mean transplacental p.d. of 6-3 mV (fetus negative) and the result expressed relative to the corrected PS-product for radioactive Na from mother to fetus as unity. Measures of relative permeabilities in both directions are thus obtained, Table 4. The placenta exhibits considerable selectivity with respect to transport of the four cations studied. For transport both towards and away from the fetus, the selectivity is Rb K > Na > Li. Whilst the sequences are the same for each direction, the sizes of the ratios differ more markedly for transport from mother to fetus. The ratios suggest net active movement of Na from mother to fetus, and of net active transport of Li and K in the reverse direction. Such a distribution of active transport is compatible with the pressure of a dominant conventional Na-K pump in the placenta, directed with its active Na component towards the mother (Atulc & Avihovec, 1977). -
Significance of ouabain effects The question may be asked as to whether the changes in K and Rb movement associated with ouabain in the perfusion fluid are due to specific inhibition of transport, since even at the lower concentration of 1O-5 M this compound has some influence on the placental circulations. The slow rise in perfusion pressure indicates a proportionate increase in vascular resistance on the umbilical side, but it developed more slowly than the effect on 42K and "Rb extraction and on net K movement. Whilst fractional entry of 3H20 was generally somewhat reduced by 10-5 M-ouabain, the effect was variable and in the experiments on 42K and 86Rb extraction very small compared with that on the cation extractions. The time course of the umbilical vascular effect and the small relative size of the maternal effect, taken together with the striking absence of any reduction in sodium movement in either direction, due to 10-5 m-ouabain strengthen the belief that the inhibitor is also acting specifically on
PLACENTAL CATION TRANSPORT 55 potassium and rubidium transport. At 5 x 10-5 M, ouabain caused a profound decrease in maternal blood flow, but it is perhaps significant that fractional entry of 86Rb was more markedly reduced. Model for transplacental transport in guinea-pig Experiments on the transport of polar non-electrolytes of differing molecular weight across the guinea-pig placenta in both the intact and the perfused in situ conditions (Faber, 1973; M. W. B. Bradbury, V. M. France & R. Hedley, unpublished observations) indicate a water-filled transplacental route. There is a direct relation between transplacental permeability and free diffusion coefficient in water. Such observations imply the presence of effective water-filled channels of large diameter, 10 nm or more. A significant fraction of cation transport may follow this pathway, but the degree of cation selectivity proves that a more specific route must also exist. A high proportion of the transplacental fluxes of 42K and 86Rb might be transcellular. The active components of these fluxes would involve some ouabain-dependent potassium pumping at the fetal-facing membrane of the syncytiotrophoblast. The evidence for such a pump at this membrane is inhibition by ouabain of 42K and 86Rb extraction from the perfusion fluid; blocking of 42K and 86Rb accumulation from the perfusion fluid by ouabain; and induction by ouabain of a net flux of K into the perfusion fluid. The less definitive evidence for a K pump directed to the opposite sense at the maternal-facing membrane of the trophoblast is greater accumulation of 42K and 86Rb by the placenta from its maternal rather than its fetal side, greater reduction of fractional entry of 86Rb than of 3H20 by 5 x 10-5mi-ouabain and histochemical demonstration of maximum density of Na-K-adenosine triphosphatase in relation to the maternal microvilli (Firth & Farr, 1977). The negative potential may then be tentatively attributed, as was done by gtulc & Svihovec (1973), to dominant electrogenic pumping of sodium ions at the maternal facing membrane of the syncytiotrophoblast. Since the low Na fluid must deplete intracellular Na, the active electrogenicity will be reduced. Since ouabain in the perfusion fluid must primarily affect the pump in the fetal facing membrane, the potential will be unchanged or even increased. Although the presence of fixed waterfilled channels might have escaped detection in electron micrographs, such low resistance pathways for current flow would hardly support a transplacental potential difference of the size measured. Opening and closing vesicles or tubules would have a higher resistance. This work was supported by an M.R.C. project grant. REFERENCES
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