R.D.H. Boyd

Amino acid (system A) transporter activity in microvillous membrane vesicles from the placentas of appropriate and small for gestational age babies

ABSTRACT: 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.

Increased maternofetal calcium flux in parathyroid hormone‐related protein‐null mice

The role of parathyroid hormone‐related protein (PTHrP) in fetal calcium homeostasis and placental calcium transport was examined in mice homozygous for the deletion of the PTHrP gene (PTHrP−/− null; NL) compared to PTHrP+/+ (wild‐type; WT) and PTHrP+/− (heterozygous; HZ) littermates. Fetal blood ionized calcium was significantly reduced in NL fetuses compared to WT and HZ groups at 18 days of pregnancy (dp) with abolition of the fetomaternal calcium gradient. In situ placental perfusion of the umbilical circulation at 18 dp was used to measure unidirectional clearance of 45Ca across the placenta in maternofetal (CaKmf) and fetoplacental (CaKfp) directions; CaKfp was < 5% of CaKmf for all genotypes. At 18 dp, CaKmf across perfused placenta and intact placenta (CaKmf(intact)) were similar and concordant with net calcium accretion rates in vivo. CaKmf was significantly raised in NL fetuses compared to WT and HZ littermates. Calcium accretion was significantly elevated in NL fetuses by 19 dp. Placental calbindin‐D9K expression in NL fetuses was marginally enhanced (P < 0.07) but expression of TRPV6/ECaC2 and plasma membrane Ca2+‐ATPase (PMCA) isoforms 1 and 4 were unaltered. We conclude that PTHrP is an important regulator of fetal calcium homeostasis with its predominant effect being on unidirectional maternofetal transfer, probably mediated by modifying placental calbindin‐D9K expression. In situ perfusion of mouse placenta is a robust methodology for allowing detailed dissection of placental transfer mechanisms in genetically modified mice.

Effect of hyposmotic challenge on microvillous membrane potential in isolated human placental villi

This study examined the effect of hyposmotic solutions on the syncytiotrophoblast microvillous membrane potential ( E m) in mature intermediate villi isolated from term human placentas. When villi were exposed to a control solution (280 mosmol/kgH2O; 116 mM NaCl) and then to either a 138-hyposmotic (138 mosmol/kgH2O; 37 mM NaCl) or 170-hyposmotic (170 mosmol/kgH2O; 55 mM NaCl) solution, there was a significant hyperpolarization of E m (-5.1 ± 1.5 mV, P < 0.01 and -5.0 ± 0.5 mV, P < 0.001, respectively; n = 10), which was reversible on removal of the hyposmotic stimulus. Low-NaCl (37 and 55 mM) solutions made isosmotic with control (i.e., 280 mosmol/kgH2O) by addition of raffinose did not significantly alter E m, suggesting that reducing NaCl concentration per se had no effect on E m. Exposure to 170-hyposmotic solution in the presence of 5 mM BaCl2 depolarized E m by +4.1 ± 0.7 mV ( P < 0.001, n = 6); BaCl2 similarly depolarized E m when added in control solution (+5.6 ± 1.1 mV, n= 5). Exposure to 170-hyposmotic solution containing 1 mM DIDS hyperpolarized E mby -9.0 ± 1.7 mV ( P < 0.001, n = 5). This degree of hyperpolarization was significantly greater than that observed in hyposmotic solution alone ( P < 0.01) but was not different from the hyperpolarization when DIDS was added to control solution (-7.4 ± 0.2 mV, n = 6). We conclude 1) that Ba2+-sensitive K+ conductances and DIDS-sensitive anion conductances contribute to the resting potential of the syncytiotrophoblast microvillous membrane and 2) that the syncytiotrophoblast microvillous membrane responds to a hyposmotic stimulus by activating both Ba2+-sensitive K+ and DIDS-sensitive anion conductances.

Microvillous membrane potential (Em) in villi from first trimester human placenta: comparison toEm at term

The microvillous membrane (MVM) potential ( E m) of first trimester human placental villi was measured and compared with that in villi from term human placentas. The median E m in first trimester villi (-28 mV) was significantly more negative than that at term (-21 mV; P < 0.001). The median E m measured in villi from early ( weeks 6-11) first trimester (-32 mV) was significantly more negative than that in late ( weeks 12 and 13) first trimester villi (-24 mV; P < 0.001). Elevating extracellular KCl concentration induced a significant depolarization of E m in both first trimester and term villi ( P < 0.05 and P < 0.001, respectively). The magnitude of this depolarization was greater in first trimester than at term, indicating that the ion conductance of the MVM changes with gestation. Exposure to ouabain induced a significant depolarization of E m (3 mV: P < 0.05) in first trimester villi but had little effect at term. These results suggest that microvillous membrane electrophysiology changes with placental development. An alteration in the relative K+:Cl-conductance of the MVM is likely to be a major contributor to the change in the magnitude of E m.

CHAPTER 52 – Placental Transfer

The placenta is the organ in which the two microcirculations, maternal and fetal, are opposed and across which most, but not all transfer takes place. For any permeant to cross the placenta, it has to traverse, or bypass, several lipid bilayer barriers and several fluid compartments. For some, transfer may also involve the release from or uptake by blood cells, plasma complexes, or carrier proteins; or metabolic interconversion within the placenta. The flux (rate of transfer) through each of the barriers is a function of both the ease of movement through it (its permeability) and on the difference in relevant electrical, chemical, hydrostatic, or thermal driving force across it. Movement through the fluid compartments is likely to be a function of diffusion and convection and is usually assumed not to be rate limiting. Overall, the rate will depend on the perfused surface area of the placenta. The capacity of the placenta to transfer solutes and water has to be sufficient to allow fetal growth. Moreover, it is likely that the transfer capacity of the placenta also changes during the course of pregnancy.

Fetal and maternal blood volumes in shed human placentae: discrepant results comparing morphometry to haemoglobin content.

The maternal blood volume (MBV) and fetal blood volume (FBV) of shed human placentae delivered by caesarean section at term were measured using a morphometric technique and from the placental content of adult and fetal haemoglobin. MBV was 35.3 +/- 1.5 (s.e.m.) per cent by the former and 11.0 +/- 1.5 per cent by the latter technique. FBV was 11.0 +/- 0.7 and 9.0 +/- 0.6 per cent respectively (n = 6). Measurement of the dimensions of individual villi initially photomicrographed in 0.9 per cent NaCl and subsequently re-photographed in fixative suggested that individual villi shrank to 0.7 of their initial volume during fixation. It is suggested that morphometric measurement of MBV may lead to approximately a threefold overestimate because of relative MBV expansion and villous tissue shrinkage during the process of fixation without alteration in overall placental volume.

Placental water content and distribution

Summary The percentage of total placental water (%H 2 O (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 51 CrEDTA space after incubation of placental fragments. Normal ranges (mean± sd ) after term vaginal delivery were: H 2 O (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%. %H 2 O (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 %H 2 O (T) above the mean +2 sd of term placentas and five of 17 IUGR placentas were below the mean −2 sd . The remaining placentas following maternal pre-eclampsia, hypertension, or diabetes had no apparent alteration in %H 2 O (T) . A blind histological diagnosis of ‘true’ oedema was associated with both a significantly high %IW and %H 2 O (T) . Perhaps this is due to alteration in placental cell volume regulation in certain situations.

Site of catecholamine modulation of feto-maternal electric potential difference in the pig

Feto‐maternal vascular (PD(F‐M)) and amniotic maternal (PD(A‐M)) potential differences were measured simultaneously on seven occasions in six conscious pigs of 100–106 days gestation. Resting values of PD(F‐M) and PD(A‐M) were not significantly different although the range was wide. Fetal intravascular injection of 20 microg adrenaline, but not of saline, was associated with a prompt reversible change, of equal magnitude, in both PD(F‐M) and PD(A‐M). In some experiments polarity was reversed. Feto‐amniotic potential difference did not change. There was no change in fetal plasma K+ and Na+ concentrations. Because of the simultaneous and equal alterations in PD(F‐M) and PD(A‐M) following adrenaline and the anatomical configuration of the pig conceptus, we conclude that the catecholamine modifiable component of PD(F‐M) is generated by electrogenesis in the pig placenta, probably by its chorionic (trophoblastic) cell layer.