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The effect of maternal diabetes mellitus on placental unidirectional maternofetal flux of calcium (Ca) and magnesium (Mg), calbindin9K mRNA expression, and net fetal Ca and Mg accretion has been investigated using control (C), untreated diabetic (D(O)) and insulin-treated diabetic (DI) rats. Unidirectional maternofetal flux of Ca in the D(O) group was 61 and 63% of the value of the C and DI groups; unidirectional maternofetal flux of magnesium in the D(O) group was 79 and 66% of the value in the C and DI groups. Fetal Ca and Mg content (mmol; mean +/- SEM) was also significantly lower in the D(O) group compared with the other two groups (0.111 +/- 0.004 versus 0.153 +/- 0.008 in C and 0.168 +/- 0.007 in DI, p < 0.01 D(O) versus C and DI for Ca; and 0.021 +/- 0.001 versus 0.027 +/- 0.001 in C and 0.031 +/- 0.001 in DI, p < 0.01 D(O) versus C and DI for Mg). However, only Ca content was significantly lower in the D(O) group when normalized to fetal ash weight. Densitometric analysis of autoradiograms after Northern hybridization with cDNA probes demonstrated that the placental calbindin9K/cyclophilin mRNA ratio was 11- to 12-fold lower in the D(O) group compared with the C and DI groups. Collectively, the data suggest that untreated maternal diabetes mellitus reduces fetal Ca and Mg accretion by an effect on the expression of placental transport components involved in the maternofetal transfer of these cations.

The percentage of total placental water (%H2O(T)), maternal (%MBV) and fetal (%FBV) blood volumes, non-vascular extracellular (%EW) and intracellular (%IW) water, and villous histology were studied in placentas from 12 normal term pregnancies after a normal vaginal delivery, 19 caesarean sections at term after a normal pregnancy and history of a previous caesarean section and 47 caesarean sections at term or preterm due to pregnancy complications. Values were derived from change in placental dry weight, maternal and fetal haemoglobin content and 51CrEDTA space after incubation of placental fragments. Normal ranges (mean +/- SD) after term vaginal delivery were: H2O(T) 83.9 +/- 0.2%, MBV 10.9 +/- 0.2%, FBV 7.4 +/- 0.9%, EW 57.3 +/- 1.3% and IW 11.2 +/- 0.6%. %H2O(T) was higher after caesarean section; other measurements were not affected. There were no differences between placentas after 33-37 and after 38-42 weeks gestation. Three of eight placentas after rhesus incompatibility had %H2O(T) above the mean +2SD of term placentas and five of 17 IUGR placentas were below the mean -2SD. The remaining placentas following maternal pre-eclampsia, hypertension, or diabetes had no apparent alteration in %H2O(T). A blind histological diagnosis of 'true' oedema was associated with both a significantly high %IW and %H2O(T). Perhaps this is due to alteration in placental cell volume regulation in certain situations.

Unidirectional uptake of chloride by microvillous membrane vesicles prepared from human term placentas was studied over a range of membrane potentials in the presence and absence of chloride transport inhibitors alone and in combination at maximally effective concentrations. At 0 mV, inhibition of chloride uptake by 0.1 mM DIDS, 0.5 mM DPC, and 0.5 mM DPC plus 0.1 mM DIDS was similar, suggesting a common action upon an anion exchanger; neither 0.1 mM furosemide nor 0.1 mM bumetanide alone had any effect. An inside-positive membrane potential was created by imposing an inwardly directed potassium ion gradient in the presence of valinomycin. Total chloride uptake increased with increasing membrane potential (0, 4.6, 17.3, 25.8 and 32.0 mV). The inhibition of uptake by DPC and DPC/DIDS increased with the membrane potential. The effect of DPC compared to DPC/DIDS was significantly different at 4.6, 17.3 and 25.8 mV, suggesting a degree of additivity of inhibition. Neither furosemide nor bumetanide had any effect at any potential. There was a significant increase in inhibition due to DIDS alone until the membrane potential reached 25.8 mV. But there was no significant difference between the level of inhibition at 32 mV as compared to 0 mV, providing evidence of a DIDS-sensitive conductance similar to that previously seen in patch clamp studies. We suggest that uptake of chloride across the microvillous membrane of the human placenta may be by at least three different pathways; an electroneutral, DIDS-sensitive anion exchanger, a DPC-sensitive chloride conductance and a DIDS-sensitive chloride conductance.

Although a number of causes of poor fetal growth are known, the involvement of placental transport proteins in the etiology of growth retardation is not understood. The aim of this study was to investigate the activity of the system A amino acid transporter and the Na+/H+ exchanger in vesicles isolated from the microvillous membrane of the syncytiotrophoblast of placentas of appropriate and small for gestational age babies. There were no biochemical differences between the membranes from the two groups of placentas, and there was no difference in the activity of the Na+/H+ exchanger. The initial rate of uptake of methylaminoisobutyric acid (a nonmetabolizable amino acid analogue) was 63% lower in vesicles from placentas of small for gestational age babies. Kinetic analysis of the system A transporter (utilized by methylaminoisobutyric acid) showed that the Vmax in the vesicles from placentas of small for gestational age babies (0.24 +/- 0.03 nmol/mg protein/30 s, n = 5) was significantly lower than that in vesicles from placentas of appropriate for gestational age babies (0.64 +/- 0.09 nmol/mg protein/30 s, n = 4, p < 0.001), whereas the Km was not different between the two groups. It is concluded that there is an abnormality of system A amino acid transporter function in placentas of small for gestational age babies.

1. In order to investigate mechanisms of Na+ transfer, the unidirectional maternal-fetal clearance (Kmf) of 22Na+ and of 51Cr-EDTA (a marker of paracellular diffusion) was measured across the intact or umbilically or dually perfused placenta of the anaesthetized rat. 2. The Kmf of 22Na+ in the intact preparation (18.5 +/- 2.7 microliters min-1, mean +/- S.D., n = 105 placentas) exceeded that of 51Cr-EDTA in the same experiments (1.4 +/- 0.3 microliters min-1) by more than ten times, whereas the difference in their diffusion coefficients in water was only 2-fold. In the perfused preparations the difference in the Kmf values was 6-fold. 3. Assuming that a simple model of paracellular diffusion through wide pores was one component of transfer, the Kmf of 51Cr-EDTA and the diffusion coefficients were used to calculate a component of 22Na+ clearance (Kmf,residual) and of Na+ flux (Jmf,residual) across the perfused placentas which could not be accounted for by transfer through the paracellular route. 4. Kmf,residual of 22Na+ across the dually perfused placenta was significantly lower when temperature was reduced, the temperature quotient (Q10) of the transfer being about 2. Kmf,residual was also significantly lower when 0.1 mM ouabain was perfused on the fetal side. Jmf,residual exhibited saturation kinetics characterized by an apparent Michaelis constant (Km) of 90 mM. Kmf,residual was not influenced by 0.5 mM frusemide, 0.5 mM amiloride or by 0.5 mM hydrochlorothiazide administered to the maternal side. It was significantly increased by 1 mM alanine on the maternal side suggesting that the coupled transfer of Na+ and amino acids may contribute significantly to the maternal-fetal flux of Na+. 5. These observations suggest that most (80%) of the maternal-fetal flux of Na+ across the rat placenta is effected by active transcellular transport. This transport involves passive entry of Na+ into the trophoblast from the maternal side by a largely unknown saturable mechanism and active extrusion of Na+ from trophoblast to the fetal side by Na(+)-K(+)-ATPase.

The purpose of this study was to directly investigate the mechanisms of K+ transfer across the rat placenta, which was isolated and perfused through both its maternal and fetal circulations. Unidirectional maternofetal (Kmf) and fetomaternal (Kfm) clearances for 42K, 51Cr-labeled EDTA (used as a diffusion-limited paracellular marker), and 3H2O (used as a flow-limited marker) were respectively 232 +/- 36, 12 +/- 4, and 1,020 +/- 260 (mf) and 96 +/- 26, 18 +/- 6, and 737 +/- 176 (fm) microliters.min-1 x g placenta-1. Calculated K+ fluxes were asymmetric, being 0.75 +/- 0.12 and 0.41 +/- 0.12 mumol.min-1 x g placenta-1 for maternofetal and fetomaternal, respectively (mean +/- SE, n = 6; P < 0.01, paired t test). Although Kmf for 3H2O was 28% higher than Kfm, this could not completely account for the asymmetry in K+ fluxes. Kmf for 42K was 12-70 times higher than that for 51Cr-EDTA (presumed to be a paracellular marker), although its diffusion coefficient is only 2.5 times higher. An apparent Michaelis constant (Km) of 11.0 +/- 2.4 mM and maximum velocity (Vmax) of 3.8 +/- 0.33 mumol.min-1 x g placenta-1 was calculated by Michaelis-Menten analysis of the transcellular component of maternofetal flux (Jmf) for K+. Ouabain or barium (1 mM in maternal and fetal perfusate) reduced Kmf for 42K from 250 +/- 38 to 76 +/- 13 microliters.min-1 x g placenta-1 (n = 4; P < 0.01) and from 358 +/- 31 to 106 +/- 18 microliters.min-1 x g placenta-1 (n = 5; P < 0.001). Neither drug had any effect on Kmf for 51Cr-EDTA or 3H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Single mature intermediate villi from term placentas after normal gestation and vaginal or cesarian delivery were identified microscopically and mounted in a bath for conventional micro-electrode studies. With the application of strict selection criteria, the following observations on electrical potential difference (PD) were made. 1) With the tissue bathed in Earle's medium (37 degrees C) the PD across the syncytiotrophoblast microvillus membrane with respect to the bath was not normally distributed. The median PD was -22 mV (range -12 to -60 mV, n = 200). This fell to -6 mV after prior incubation for 4 h with cyanide (3 mM) or iodoacetate (2 mM) but was not altered by short-term application of these agents or by 0.1 mM ouabain. Substituting Na+ in Earle's medium with choline had no effect on PD, but replacing Cl- with gluconate caused a depolarization of 6 mV (P < 0.002). Increasing KCl in the bath fluid revealed an apparently low microvillus membrane K+ conductance. The low microvillus membrane PD may reflect a low Na(+)-K(+)-ATPase activity and/or a low membrane permeability to K+. 2) The transtrophoblast PD measured by insertion and withdrawal of the electrode was significantly different from zero [P < 0.003, median -3 mV (range 0 to -15 mV), n = 11]; and PD measured by insertion of the electrode into the villus core and beyond was -6 mV (significantly different from zero P < 0.003, range -2.5 to -10 mV, n = 6). If a similar PD were to exist in vivo, it could have a significant influence on ion transport across the placenta at term.

Maternal and fetal magnesium homeostasis is reviewed. Current evidence suggests that pregnancy-associated growth is unlikely to cause maternal magnesium deficiency and that the case for magnesium supplementation during pregnancy is unproven. Similarly, data do not support the use of magnesium in prevention of preterm labor or its preferential use in tocolysis. Magnesium might have a role to play in the prevention/treatment of eclamptic seizures. Hypomagnesaemia occurs in poorly controlled diabetic pregnancy and may be part of the cause of the hypomagnesaemia in the infants of such mothers. Fetal magnesium homeostasis is poorly understood as is the mechanism of placental transfer. Data from the rat suggest that the bulk of maternofetal placental magnesium transfer occurs via a transcellular route utilising a Na+/Mg2+ exchanger and that maternofetal flux of magnesium is reduced in the presence of maternal diabetes mellitus. Further study of diabetic pregnancy will increase our understanding of magnesium homeostasis both in normal and abnormal pregnancy.

Physiological data indicate that both a transcellular and a paracellular pathway are available for transfer across the human placenta but the morphological correlate of the latter is uncertain. We measured the permeability of the dually perfused human placental cotyledon to the predominantly cationic protein horseradish peroxidase (MW 40,000), to the neutral polymer 14C-dextran (MW 50-70,000) and to the extracellular space marker creatinine (MW 113). Following fixation and cytochemistry, we used brightfield microscopy to localize peroxidase reaction product within the same tissue. Steady state unidirectional maternofetal clearance (Kmf) for the peroxidase (0.90 +/- 0.27 microliters/min/g, n = 9) was not significantly different from that for 14C-dextran (0.95 +/- 0.07 microliters/min/g, n = 3) suggesting that charge does not markedly influence peroxidase permeability. The Kmf for creatinine was 13.1 +/- 2.5 microliters/min/g (n = 9); these permeability data are similar to those reported for the placenta in vivo. Microscopically, peroxidase reaction product was localized to the microvillous surface of the syncytiotrophoblast of most villi and to the trophoblastic basement membrane and connective tissue cells of the villous core in a few villi. The reaction product was also associated with fibrin-containing deposits attached to the villous core at sites of discontinuity of the syncytial epithelium. The staining pattern within the deposits was consistent with a diffusion gradient of the peroxidase. These fibrin-containing deposits at discontinuities in the syncytiotrophoblast may provide one paracellular route for peroxidase diffusion from the intervillous space into the villous core.

The unidirectional maternofetal clearance (Kmf) of 45Ca was measured across the rat placenta over the last one-third of gestation. Kmf for 45Ca normalized to its diffusion coefficient in water (Kmf/Dw) increased 72-fold between days 15 and 22 of gestation from 3.5 +/- 0.3 to 253.1 +/- 22.0 cm/g placenta, respectively. At 15 and 18 days of gestation, Kmf/Dw for 45Ca was similar to Kmf/Dw for the paracellular marker [14C]mannitol, but at 21 and 22 days of gestation, Kmf/Dw for 45Ca was significantly higher than Kmf/Dw for [14C]mannitol, indicating that an additional route of transfer, other than diffusion, becomes available to calcium during this period. Northern hybridization analysis demonstrated that rat placental calbindin9K-to-beta-actin mRNA ratio increased 135-fold between 15 and 22 days of gestation and was temporally associated with the gestational increase in Kmf/Dw for 45Ca. In contrast, rat placental Ca(2+)-ATPase-to-beta-actin mRNA ratio increased only two- to threefold over the same gestational period and did not mirror the gestational changes in calcium clearance. These trends suggest that the expression of placental calbindin9K, but not Ca(2+)-ATPase, may be rate limiting to placental calcium transport in the rat.

Potassium (K+) fluxes across the placenta of rats, at 21 days gestation, fed a low K+ diet or a control diet were studied. The rats on the low K+ diet had a significantly (P less than 0.001) lower arterial plasma K+ concentration compared to those on the control diet (1.95 +/- 0.12 and 2.93 +/- 0.06 mmol/l respectively; mean +/- s.e., n = 17). Fetal umbilical arterial plasma K+ concentration was unaltered in maternal hypokalaemia and was significantly (P less than 0.001) higher than that of maternal plasma (4.58 +/- 0.15 and 4.66 +/- 0.12 mmol/l in low K+ and control groups respectively). Net K+ flux across the placentas (as measured by fetal accretion between days 20 and 21 of gestation) of hypokalaemic mothers (0.106 +/- 0.02 mumol/min/g placenta, n = 6) was not different to that in controls (0.104 +/- 0.01 mumol/min/g placenta, n = 8). Unidirectional maternofetal flux (Jmf) across the placentas, measured as the accumulation of 42K in the fetuses after injection of the radioisotope into maternal blood, was also not significantly different between the hypokalaemic and control mothers (0.5 +/- 0.08 mumol/min/g placenta, n = 8 versus 0.63 +/- 0.06 mumol/min/g placenta, n = 7, respectively). However, measurement of Jmf by perfusion of the placentas through their fetal circulations yielded a higher value than the accumulation method and in this analysis was significantly (P less than 0.02) lower in the low K+ than in the control group (0.75 +/- 0.10 mumol/min/g placenta, n = 11, and 1.27 +/- 0.14 mumol/min/g placenta, n = 9, respectively). These results show that net placental K+ fluxes are unaltered during maternal hypokalaemia but suggest that unidirectional maternofetal fluxes may be reduced.

To investigate how the passive permeability of the rat placenta changes during the last third of gestation we measured the unidirectional maternofetal clearance (Kmf) of three polar nonelectrolytes. We found that Kmf (microliters.min-1.g placenta-1) for all three tracers increased between days 15 and 22 (term is 23 days). The increase was less for the largest tracer, [14C]inulin (3.5-fold increase from 0.5 +/- 0.1 to 1.6 +/- 0.1 microliters.min-1.g placenta-1), than for the smaller tracers, [14C]mannitol (7-fold increase from 1.6 +/- 0.1 to 10.9 +/- 0.4 microliters.min-1.g placenta-1) and 51Cr-labeled EDTA (9.5-fold increase from 0.8 +/- 0.04 to 7.6 +/- 0.7 microliters.min-1.g placenta-1). These data suggest that whilst the overall permeability of the rat placenta increases during this period of pregnancy, restriction to the diffusion of large (greater than or equal to 1.5 nm molecular radius) polar nonelectrolytes also increases.

Lipase activities were measured at pH 4 and pH 8 in the placentas of rats made diabetic by streptozotocin treatment and also in the placentas of women classified as having 1) impaired glucose tolerance or type 2 diabetes, 2) type 1 diabetes with no associated vascular complication, and 3) type 1 diabetes with associated vascular disease. In both sets of experiments, the placentas were compared with normal control groups. The placental lipase activity measured at pH 8 was not significantly different in either streptozotocin-treated rats or impaired glucose tolerance/diabetic women as compared with controls, whereas the lipase activity measured at pH 4 increased significantly as compared with controls in both species. Furthermore, in the women there was a significant correlation between placental lipase activity at pH 4 and birth weight in impaired glucose tolerance/type 2 diabetes. It is suggested that the increased placental lipase activity may contribute to the increased fetal weight in human diabetic pregnancy, by contributing to the increased fat transfer across the placenta from mother to fetus.

Placentas of anesthetized rats were perfused in situ on the fetal side to study mechanisms of Mg2+ transport. The perfusate was a Mg(2+)-free Krebs-Ringer, and the unidirectional transfer of Mg2+ from maternal plasma to this Ringer was compared with that of 45Ca and 51Cr-EDTA, the latter being employed as a paracellular diffusional marker. Placental perfusion with amiloride (0.5 mM) or ouabain (1 mM) both rapidly (4 min) reduced maternal-fetal clearance (Kmf) for Mg2+ but had no effect on Kmf for 45Ca. In contrast, perfusion of the carbonic anhydrase inhibitor acetazolamide (1 mM) did not affect Kmf for Mg2+ or 45Ca. Placental perfusion with a Na+-free Ringer reduced Kmf for both Mg2+ and 45Ca, although the latter response was delayed. Kmf for 51Cr-EDTA was increased by amiloride and was unaffected by perfusion of ouabain, acetazolamide, or Na+-free Ringer, indicating that the effects of these treatments on Kmf of Mg2+ do not reflect nonspecific effects on placental permeability. These data suggest that maternal-fetal transfer of Mg2+ across the perfused rat placenta is Na+ dependent.

Two human parathyroid hormone-related protein (hPTHrP) fragments were tested for effects on maternofetal transfer of 45Ca and Mg across the in-situ perfused rat placenta at 21 days of gestation (term = 23 days). The fetal placental circulation was perfused with a Mg-free Krebs-Ringer solution and the unidirectional maternofetal clearance (Kmf) of 45Ca and Mg compared with that of 51Cr-EDTA, the latter being employed as a paracellular diffusional marker. Placental perfusion with hPTHrP(1-34) (100 ng/ml) or hPTHrP(75-86)amide (50 ng/ml) did not significantly alter the Kmf of 45Ca or that of Mg. In separate rats, however, hPTHrP(1-34) but not hPTHrP(75-86)amide stimulated marked placental cyclic AMP (cAMP) release, the peak response of 63 +/- 7 pmol/min occurring 10 min after the beginning of the peptide perfusion. A lower dose of hPTHrP(1-34) (4 ng/ml) produced a similar peak release of cAMP, as did [Nle8,21, Tyr34]-rPTH(1-34)amide (4 ng/ml) and the adenylate cyclase agonist forskolin (17 mumol/l). Forskolin also rapidly increased the Kmf of 45Ca but not that of Mg or 51Cr-EDTA. The present study indicates that hPTHrP does not acutely affect maternofetal transfer of Ca or Mg across the perfused rat placenta. The data also question the role played by cAMP in the stimulatory actions of forskolin on placental Ca transport.

8-Bromo-adenosine (8-Br-adenosine) stimulates CG production in the JAr choriocarcinoma cell line. Because production of the alpha and beta CG subunits is coupled to the differentiation state of the trophoblasts, we compared the effect of adenosine on CG synthesis in the choriocarcinoma cell line with that in cytotrophoblast cells isolated from normal human term placenta. 8-Br-adenosine stimulated CG production by 5- to 6-fold in both cell types, whereas 8-Br-guanosine had no effect, demonstrating specificity for adenine-containing derivatives. The ratio of CG alpha to CG beta production in JAr cells was 2:1 in the absence or presence of 8-Br-adenosine, whereas in cytotrophoblasts the ratio was 14:1 in controls, but decreased to 3:1 with 8-Br-adenosine. The latter is attributed to an enhanced production of the beta-sub-unit. Immunocytochemistry revealed that CG alpha and CG beta were only expressed in about 4% of JAr cells, and 8-Br-adenosine stimulated the number of positive cells 2- to 4-fold. Since 8-Br-adenosine also inhibited the division of JAr cells, the data suggest that it stimulates CG expression by promoting exit of a population of the cells from the cell cycle. In nondividing placenta-derived cytotrophoblasts, 8-Br-adenosine affects a later step in the differentiation pathway, resulting in a greater effect on the beta- than the alpha-subunit. Thus, our data further support the hypothesis that regulation of CG biosynthesis is linked to differentiation of the trophoblast.

Lipase activities measured at pH 4 and 8 were determined in the placentas of rats subjected to undernutrition or uterine artery ligation. The fetal lipid content following maternal undernutrition was also determined. The placental lipase activity (per gram placenta) measured at pH 8 (placental lipoprotein lipase) remained unchanged, whilst the activity of the intracellular lipase (per gram placenta) measured at pH 4 was significantly less than in the control group (21% lower in maternal undernutrition and 16% lower after uterine artery ligation). Although placentas and fetuses were significantly lighter in both situations as compared to controls (well-nourished or sham operated, respectively), fetal lipid content in maternal undernutrition was only reduced to a degree concomitant with the decrease in fetal weight. We therefore conclude that the intracellular lipase may not have an important role in the provision of free fatty acids in undernutrition.

Cytochemistry revealed that alkaline phosphatase was localized predominantly to the maternal facing plasma membrane of syncytiotrophoblast layer II in the haemotrichorial rat placenta at term. Plasma membrane vesicles prepared from term rat placenta by homogenization, treatment with MgCl2, and differential centrifugation were enriched 14-fold in alkaline phosphatase activity as compared to homogenate. These vesicles were mainly oriented right side out, as shown by a lack of effect of saponin treatment on alkaline phosphatase activity. Na+ uptake into the vesicles under equilibrium exchange conditions was significantly stimulated (P less than 0.01) nearly threefold in the presence of an outwardly directed H+ gradient as compared to no gradient. The stimulation by a H+ gradient was abolished in the presence of 0.5 mM amiloride. Measurements with the pH-sensitive dye BCECF in the absence of Na+ showed that 95.2 +/- 0.6 per cent of a 1.2 pH unit H+ gradient was dissipated from the vesicles in 2 min, but the remaining gradient was maintained for up to 15 min. These experiments therefore provide evidence that vesicles derived mainly from the maternal facing plasma membrane of syncytiotrophoblast layer II of the rat placenta possess a Na+/H+ exchanger.

Mechanisms of maternofetal Mg transfer have been investigated across the in situ perfused rat placenta at 21 d gestation (term = 23 d). The fetal placental circulation was perfused with Mg-free Krebs-Ringer solution and clearance of Mg from maternal plasma across the placenta [unidirectional maternofetal clearance (Kmf) Mg] compared with that for 45Ca and 51Cr-EDTA, the latter being used as a diffusional marker. Because diffusion coefficients determined for these solutes were similar (6.8-7.6 X 10(-6) cm2.sec-1), greater Kmf values determined for Mg and 45Ca (mean +/- SD: 26.7 +/- 9.2 and 93.1 +/- 29.8 microL.min-1.g-1 placenta, respectively) compared to 51Cr-EDTA (3.2 +/- 0.9 microL.min-1.g-1) suggest that maternofetal transfer of these cations occurs by mechanisms in addition to diffusion. Kmf Mg was also greater than Kmf 51Cr-EDTA when measured across the dually perfused rat placenta, in which the maternal uterine artery was additionally perfused with Mg-containing (0.5 mmol.L-1) Krebs-Ringer solution. Decreasing the Mg concentration in the maternal perfusate by 90% reduced Mg appearance in the fetal perfusate by 87% within 8 min; this suggests that Kmf Mg across the in situ perfused placenta largely reflects Mg transfer from maternal plasma and not simply elution of a placental Mg pool. Addition of KCN (1 mmol.L-1) to the fetal perfusate or lowering perfusate temperature from 37 to 26 degrees C significantly reduced Kmf Mg and Kmf 45Ca across the in situ perfused placenta. In contrast, Kmf 51Cr-EDTA was increased by KCN and unaffected by temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of ultrasound on placental transfer in the rat has been measured in vivo. At 15, 18, 21 and 22 days of gestation one uterine horn of each anaesthetized rat was exposed, after laparatomy, to ultrasound. The control horn was effectively shielded. Radio-labelled tracers were then injected into the maternal circulation via a jugular vein cannula and fetuses removed simultaneously from both experimental and control horns at times predetermined to ensure minimal backflux from fetus to mother. Samples of maternal plasma were also taken at suitable intervals. The radioactive content of fetuses and plasma were measured and a unidirectional materno-fetal clearance value (Kmf) for each fetus calculated. It was shown that in rats whose body temperature was accurately maintained at 37 degrees C, ultrasound at the level used in the present study had no effect on placental clearance. However, in rats whose body temperature was allowed to fall to 32 degrees C, a 3 min exposure to 1.1 MHz continuous wave ultrasound at 1 W cm-2 caused a significant (p less than 0.001) decrease in the placental clearance of 22Na (by 55%) and 51Cr-EDTA (by 45%) in rats at 15 days of gestation. These effects showed no dose response and were not observed in rats at any of the other stages of gestation which we investigated. However, in rats at 21 days of gestation the clearance of 45Ca2+ was significantly (p less than 0.01) reduced (by 30%) by a similar exposure to ultrasound. It is concluded that although an interesting biological effect of ultrasound at low body temperatures has been observed, it has little relevance for the normal use of ultrasound in obstetrics.

Mean (SE) umbilical venous plasma potassium concentrations at 5.5, 10, 20, and 30 minutes after the start of laparotomy at term were 4.75 (0.15), 5.95 (0.26), 6.81 (0.23), and 7.59 (0.81) mmol/kg plasma water. Maternal peripheral venous plasma concentrations were 4.30 (0.13) before and 4.26 (0.23) mmol/kg plasma water after laparotomy. Plasma calcium concentration in maternal and cord blood did not change significantly.

A technique for the perfusion of the rat adrenal cortex is described. With tissue culture Medium 199 the preparation was responsive in terms of steroid production of both ACTH and K+ ions. Production of corticosterone and 18-hydroxydeoxycorticosterone (18-hydroxy-DOC) was stimulated by ACTH when it was administered at rates between 5 uu.'/min and 5 mu./min. Increasing the K+ ion concentration of the perfusate from 3.6 to 5.4 and 8.9 mmol/l stimulated the production of aldosterone, 18-hydroxycorticosterone and deoxycorticosterone, although not of corticosterone or 18-hydroxy-DOC. This preparation has been used to study further the mechanism of secretion of corticosterone and 18-hydroxy-DOC. Thus, production of these two steroids was measured at different perfusion flows, varying between 0.1 and 0.6ml/min, with different levels of ACTH stimulation. Corticosterone production was significantly (P less than 0.001) increased by increasing flows both under control conditions and with ACTH was administered at constant rates of 50 uu./min or 1 mu./min. Production of 18-hydroxy-DOC was not affected by flow either under control conditions or with 50 uu. ACTH/min. However, when ACTH was administered at 1 mu./min. 18-hydroxy-DOC production was also significantly (P less than 0.001) increased by flow. The results are consistent with those obtained in previous in-vitro studies and have been interpreted as suggesting that the main mechanism of corticosterone secretion is simple diffusion. In contrast, 18-hydroxy-DOC secretion, at least at sub-maximal levels of stimulation, appears to require a more complex process.

The possibility that histamine can affect both the vascular resistance and permeability of the isolated dually perfused guinea-pig placenta has been investigated. Change from control to histamine (2.7 x 10(-4)M) perfusion of the fetal circulation elicited a significant (P less than 0.01, paired 't' test) maximum increase of 1.17 +/- 0.14 (SEM) kPa in fetal perfusion pressure 3 min later, representing a 33% rise. This vasoconstriction was completely blocked by the H1 antagonist diphenhydramine (10(-4)M) but not by the H2 receptor antagonist cimetidine (10(-4)M). In the same experiments the clearance (calculated as the ratio of fetal to maternal perfusate concentration times fetal flow-rate) of a macromolecular tracer, anionic horseradish peroxidase from the maternal to fetal circulation was significantly increased (P less than 0.05, paired 't' test) when steady state (15-20 min of perfusion) values were compared, from 5.9 +/- 1.7 (SEM) microliter min-1 placenta-1 to 12.9 +/- 3.5 (SEM) microliter min-1 placenta-1 (n = 20) for control and histamine respectively. By contrast the steady state clearance (calculated as before) of a smaller hydrophilic tracer, 51Cr-EDTA, was not significantly affected, being 587 +/- 59 (SEM) microliter min-1 placenta-1 in control and 587 +/- 55 (SEM) microliter min-1 placenta-1 (n = 20) with histamine perfusion. When histamine was perfused simultaneously with an H1 or H2 antagonist there was no change in anionic horseradish peroxidase clearance. Electron microscopy of placentas perfused with histamine failed to reveal any obvious alteration in morphology or anionic horseradish peroxidase localisation as compared to placenta perfused without histamine. This study thus demonstrates that histamine may cause changes in the macromolecular permeability of the placenta as well as vasoconstriction of the placental vasculature.

The permeability of the rat placenta at 21 days' gestation (term is 23 days) has been measured with fetuses intact in situ and by perfusion of the fetal circulation. Intact measurements were made by removal of fetuses at known time intervals after injection of radioisotopes to the anaesthetized mother and measurement of their radioactivity content. Unidirectional clearance (Kmf) was calculated and found to be in proportion to the diffusion coefficient in water (Dw) for [14C]mannitol, [14C]sucrose, [51Cr]EDTA and [3H]inulin but was relatively high for 22Na and low for [125I]albumin. The high value for 22Na is partially explained by transfer across the yolk sac placenta; cautery of the vitelline vessels significantly reduced the Kmf for 22Na but not that for [51Cr]EDTA or [125I]albumin. The low [125I]albumin value is explained on the basis of restricted diffusion of this tracer. Perfused permeability measurements were made by injection of radioisotope into the anaesthetized mother and measurement of the radioactive content of fetal perfusate samples. Although the Kmf for Na+ was significantly (P less than 0.01) reduced by 25 per cent, there was no other significant difference between results with the perfused placentae compared with those from the intact placentae. Comparison of the intact data with similar measurements in guinea pig and human confirms the similar permeability properties of the haemochorial placentae as a whole; the extra trophoblast layers in the rat make no discernible difference.

The uterine glands, glandular secretions and areolar chorionic epithelium of the pig placenta in late gestation show high levels of histochemical phosphatase activity at acid pH, particularly towards aromatic monophosphates. The properties of this phosphatase allow its identification with the placental iron carrier protein, uteroferrin, rather than with lysosomal acid phosphatase. Alkaline phosphatase activity is restricted to the microvasculature on the maternal side of the placenta and is entirely distinct from uteroferrin phosphatase activity.

Three methods were used to prepare microvillus membrane vesicles from each of six human placentas. Two of these incorporated an agitation stage to preferentially remove microvilli and either Ca2+ (Method 1) or Mg2+ (Method 2) aggregation of non-microvillus membrane. The third method involved homogenisation of the tissue followed by Mg2+ aggregation of non-microvillus membrane (Method 3). Enrichment of alkaline phosphatase activity (27.6 +/- 1.9, 25.3 +/- 2.7) and ouabain binding (5.9 +/- 2.6, 5.3 +/- 2.2, respectively) was similar in vesicles prepared by Methods 1 and 2, respectively. Method 3 vesicles showed a significantly (P less than 0.01) lower alkaline phosphatase enrichment (18.1 +/- 1.2), but ouabain binding enrichment (6.3 +/- 1.3) was not different and vesicle protein recovery (mg/g placenta) was 5-fold greater. Na+ uptake in the presence of an outwardly directed proton gradient was significantly inhibited in all microvillus membrane vesicles by amiloride (0.5 mM). However, the amiloride sensitive component of Na+ uptake was 3-6-fold greater in Method 3 vesicles than in Method 1 and 2 vesicles, and showed overshoot above equilibrium in the former but not the latter. Further experiments using the pH sensitive dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein suggested that the proton gradient dissipated faster from Method 1 than from Method 3 vesicles. Thus methodological differences can have a marked effect on transport processes in microvillus membrane vesicles prepared from the human placenta.

The endothelial cell junction in guinea-pig placental capillaries consists of a continuous ribbon desmosome (zonula adherens) within which lies a particulate tight junction consisting of between one and five anastomosing strands. The intercellular space at these tight junctions is narrowed and is subdivided by junctional bars which are probably continuous with the intramembrane particle rows seen in freeze-fracture replicas of the junctions. Perfusion with lanthanum salts shows the gaps between the junctional bars to be lanthanum-filled and the entire junction to be lanthanum permeable. The estimated size of the spaces between the junctional bars is consistent with the junctional pore size indicated by previous ultrastructural tracer studies. The wider lateral intercellular space of the ribbon desmosome is spanned by more widely spaced "linkers" which may act as a coarser three-dimensional filter in series with size-limiting pores between the tight junctional bars.

Haem proteins of different molecular sizes were perfused into the foetal circulation of the guinea-pig placenta to study the permeability of the foetal endothelium. The smallest molecules tested, microperoxidase (ae 1.0 nm) and cytochrome C (ae 1.5 nm), readily penetrated the endothelium; tracer-reaction product was found in the subendothelial space of the capillaries. However, there was no uptake of these two tracers into the syncytiotrophoblast layer of the placenta. An intermediate-sized molecule, myoglobin (ae 1.7 nm) produced only a weak reaction product in the subendothelial space even when perfused at high concentration. The largest molecule tested, haemoglobin (ae 2.8 nm), did not penetrate the foetal endothelium at any of the concentrations employed. The foetal capillary endothelium thus provided a barrier to protein penetration from the foetal circulation, dependent on molecular size. There was evidence that the site of this barrier was located in the lateral intercellular spaces between the endothelial cells. The syncytiotrophoblast of this haemomonochorial placenta provided an almost absolute barrier to protein penetration from the foetal circulation. As other workers have described maternal-to-foetal transmission of proteins across this layer in the guinea-pig, a working hypothesis of the role of endothelium and syncytiotrophoblast in maternal/foetal protein exchange is discussed.

Control of maternofetal calcium transfer across the in situ perfused rat placenta at day 21 of gestation (term 23 d) was investigated in both intact fetuses and those parathyroidectomized by decapitation on day 19. Decapitation resulted in significant fetal hypocalcemia. Injection of fetuses subcutaneously through the uterine wall with 0.43 micrograms bovine (b) PTH(1-84), 20 ng 1,25(OH)2D3 or 10 microL of the appropriate diluent resulted 2 h later in a raised fetal blood ionized Ca concentration only with bPTH(1-84) in both normal and decapitated fetuses. Fetal decapitation caused a significant (p less than 0.001) fall in the clearance of 45Ca across the placenta (Kmf45Ca), which was significantly (p less than 0.05) reversed after fetal bPTH(1-84) and 1,25 dihydroxy vitamin D3 (1,25(OH)2D3) injection, but not back to normal levels. There was no effect of either hormone on Kmf45Ca in placentas from intact fetuses, or on Kmf51Cr-EDTA (used as an extracellular marker) in either group. When 4 ng/mL r[Nle8,21, Tyr34] PTH(1-34), 50 pg/mL 1,25(OH)2D3 or the appropriate diluent was perfused through placentas the only response observed was a significant (p less than 0.05) increase in Kmf45Ca with 1,25(OH)2D3 perfusion in placentas from decapitated fetuses, Kmf51Cr-EDTA being unchanged. Finally, perfusion with 10(-5) M forskolin (an activator of adenylate cyclase) stimulated Kmf45Ca in placentas from both normal and decapitated fetuses. Although there was also some effect on Kmf51Cr-EDTA in the latter, there was none in the placentas from normal fetuses, and here the effect on Kmf45Ca was dose dependent with an initial response at 10(-6) M.(ABSTRACT TRUNCATED AT 250 WORDS)

Horseradish peroxidase (HRP) was used to study macromolecule permeation into the guinea-pig placenta perfused in situ. When tissue culture medium 199 (TC 199) was used as fetal-side perfusate, the tracer reaction product was found only lining the fetal endothelium. When a longer period of perfusion with HRP in TC 199 was used, a small amount of reaction product was found in the subendothelial space and syncytiotrophoblastic vesicles, but not in maternal lacunae. In similar experiments using a Krebs bicarbonate Ringer (KRBG) as perfusate the tracer was found (i) lining the fetal endothelium, (ii) in the laternal intercellular spaces of the endothelium, (iii) in the subendothelial space, and (iv) in the maternal lacunae. It is therefore evident that the vehicle influenced the permeability of the guinea-pig placenta to horseradish peroxidase. As other studies have shown that perfusion of the fetal side with salt solution increases pore size, the results with TC 199 are regarded as more representative of the situation in the intact animal. It is therefore suggested that the fetal endothelium of the guinea-pig placenta may be largely impermeable to molecules of the size of horseradish peroxidase (4 nm) or larger.

The effect of molecular charge on protein permeability of the dually perfused guinea-pig placenta has been investigated by measurement of the permeability surface area products (PS) and observation of the ultrastructural localization of cationic horseradish peroxidase (cHRP) and anionic horseradish peroxidase (aHRP) molecules. Steady-state PS calculated from the experimental data was 0.032 +/- 0.0045 and 0.0045 +/- 0.0008 ml min-1 (mean +/- s.e.m.) for cHRP and aHRP respectively (P less than 0.01). The PS for a diffusional marker, 51Cr-ethylenediaminetetraacetic acid, showed no significant difference between the two groups. Ultrastructurally, placentae perfused with cHRP showed fewer microvilli and a dilated interstitial space compared with placentae perfused with aHRP; large vacuoles were also found in the syncytiotrophoblast in the former but not in the latter tissue. Reaction product in placentae perfused with cHRP was localized to the maternal-facing plasma membrane of the syncytiotrophoblast, in vacuoles and vesicles of the syncytiotrophoblast, and also in the basement membrane of the interstitial space, whereas placentae perfused with aHRP only had reaction product in vesicles in the syncytiotrophoblast. These results suggest that anionic sites in the guinea-pig placenta affect its permeability to charged proteins, cationic molecules inducing structural changes associated with increased permeability.

The effect of amiloride on unidirectional maternofetal clearance across the perfused placenta has been studied in rats at 21 days gestation. A bolus dose of 1 mg kg-1 followed by an infusion of 0.6 mg h-1 into the maternal circulation caused a significant (P less than 0.02) decrease in 22Na clearance by 8%, whilst having no effect on the clearance of 51Cr-EDTA; a ten times higher dose of amiloride (10 mg kg-1, 6 mg h-1) did not reduce 22Na clearance further. Similar administration of an equivalent volume of saline to control animals showed no effect on either Na+ or Cr-EDTA clearances.

Histochemical localization by Mg2+ capture methods of K+-dependent, ouabain-sensitive phosphatase activity in the pig placenta shows that strong Na+,K+-dependent adenosine triphosphatase (Na+,K+-ATPase) activity is restricted to the basal zone of the columnar epithelium covering the areolar chorionic villi. It is proposed that active Na+ absorption at this epithelium may be the source of the ouabain-sensitive, fetal-side-positive potential difference which can be measured across the placental membrane in vitro. The one-step procedure for Na+,K+-ATPase localization is unsatisfactory in this organ as any specific ATPase reaction is swamped by activity probably attributable to uteroferrin and other non-specific phosphatases.

The effect of molecular charge on the permeability to macromolecules of the fetal capillary endothelium of the guinea-pig placenta has been investigated. In a series of experiments for horseradish peroxidase (HRP) isoenzymes with different isoelectric points were perfused through the fetal circulations of guinea-pig placentae, which were then prepared for ultracytochemical localization of peroxidase activity. It was found that the two anionic HRP tracers did not penetrate the endothelial cell layer of the fetal capillaries. By contrast, both cationic HRP tracers did penetrate; reaction product was found in the subendothelial space underlying the endothelial cell layer. The route of transendothelial penetration was via the lateral intercellular spaces (LIS) between endothelial cells. In further experiments cationic and anionic ferritins were perfused. Cationic ferritins were found apparently bound in clusters to the luminal surface of the endothelial cell layer in the luminal ends of the LIS whereas very few anionic ferritin molecules were present. We conclude that molecular charge is an important determinant of macromolecule permeability through the fetal capillary endothelium of the guinea-pig placenta and may be related to the presence of anionic sites on the luminal plasma membrane of the endothelial cells.

Electrical activity generated by pieces of pig placenta, taken from anesthetized animals and mounted in Ussing chambers, has been investigated. Ten minutes after the start of voltage clamping, potential difference (PD; fetal side positive, open circuit), short circuit current (SCC), and resistance were 5.9 +/- 0.4 (SE) mV, 8.6 +/- 0.5 microA X cm-2, and 720 +/- 45 omega X cm2, respectively (n = 50). Ouabain (10(-4) M) added to the fetal side caused a maximum decline in PD and SCC from the time of addition of -3.7 +/- 0.98 mV and -3.9 +/- 1.4 microA X cm-2 (n = 6); epinephrine (10(-5) M) added to the fetal side caused increases of +1.0 +/- 0.2 mV and +4.0 +/- 1.4 microA X cm-2, respectively (n = 14). Drug concentrations for 50% maximum response for the effect of a series of adrenergic agonists on SCC were (in M) isoproterenol 1.2 +/- 0.05 X 10(-8), norepinephrine 6.1 +/- 0.3 X 10(-8), epinephrine 2.4 +/- 0.1 X 10(-7), and phenylephrine 4.7 +/- 0.2 X 10(-5), suggesting the involvement of fetally oriented beta-adrenergic receptors. Fetal epinephrine (10(-5) M) also stimulated net Na+ flux (Jnet) toward the fetal side to an extent equal to its effect on SCC. In control experiments Jnet was small but was inhibited by fetal side ouabain (10(-4) M) to produce a maternally directed Jnet, significantly different to the SCC. Replacement of Na+ by choline reduced SCC markedly but did not abolish it. In the absence of Na+, epinephrine had no effect on SCC. These results suggest that active Na+ transfer is not completely responsible for the control electrical activity of pig placenta. Epinephrine, however, modulates SCC entirely by stimulating net Na+ transfer toward the fetal side.

The extracellular volume of fresh and perfused human placenta has been measured in two sets of experiments. In the first set fragments of placenta which had been dually perfused for either 45 or 110 min as well as fragments from unperfused areas of the same placenta were incubated for 4 h in Earle's bicarbonate buffer containing 51Cr-EDTA as an extracellular marker. The 51Cr-EDTA space, as a proportion of wet weight, was 76.5 +/- (SE) 1.4 (n = 11) and 79.4 +/- 1.8% (n = 6) for the short and long perfusions, respectively; these did not significantly differ from each other or from the values for the equivalent unperfused placenta: 75.2 +/- 1.0 (n = 11) and 77.7 +/- 0.9% (n = 6). In the second set of experiments 51Cr-EDTA and 14C-inulin were perfused through the maternal and fetal circulations of the placental lobules for 1, 2, or 4 h, and simultaneously unperfused fragments from the same placenta were incubated with the markers for 1, 2, or 4 h. The 51Cr-EDTA space was found to be significantly higher than the 14C-inulin space in both perfusion and incubation determinations. Irrespective of the marker, the incubation spaces were unaltered with time and were significantly higher than the perfusion spaces at all three time points. However, the difference decreased with the length of perfusion, so that by 4 h it was less than 10%. (At 4 h incubation the 51Cr-EDTA space was 76.2 +/- 0.9% and the perfusion space 67.4 +/- 1.1%, n = 6; the 14C-inulin incubation space was 60.9 +/- 2.2% and the perfusion space 52.5 +/- 2.6%, n = 5). As there was no evidence of increasing placental permeability with time, as judged by clearance of creatinine, it was concluded that the placental extracellular volume is large irrespective of the method of measurement.

The maternofetal potential difference (PD) between catheters in maternal and fetal blood vessels has been measured in conscious sows between 97 and 107 days of gestation. The maternofetal PD was -18 +/- 4 mV (mean +/- SE, n = 13, fetus negative) on the day of surgery and -29 +/- 5 mV (n = 6) on the day after surgery. Injection of 2, 20, or 200 micrograms of epinephrine into the fetuses caused a marked rapid change in maternofetal PD such that the fetus became less negative and, in some cases, became positive with respect to the mother. The maximum change, obtained with 20 micrograms, was 19.9 +/- 5.6 mV (n = 7); measurements of fetal plasma epinephrine concentrations (using high-performance liquid chromatography) after injection of this dose gave a time 0-extrapolated concentration of 436.5 +/- 169.0 nmol/l (n = 4). Injection of 20 micrograms of the beta-agonist isoprenaline caused a maximum change in PD of 20 +/- 4 mV (n = 6); 2 mg of the alpha-agonist phenylephrine was required to produce a similar change (15 +/- 2 mV, n = 6). Injection of the beta-antagonist propranolol (1 mg) reduced the effect of 20 micrograms epinephrine by 40%. The effect of catecholamine on maternofetal PD is similar in polarity and specificity to that found for transplacental PD in vitro in the same species. There is, however, a difference between resting maternofetal and transplacental PD that remains unexplained.