Energy metabolism and glycolysis in human placental trophoblast cells during differentiation.

Energy metabolism and glycolysis of normal human term placental trophoblast in two-sided culture was investigated during differentiation from cytotrophoblast to syncytiotrophoblast, because glycogen metabolism is abnormal in several trophoblast related pregnancy diseases, including pre-eclampsia. After initial recovery of energy and cytoplasmic NADH/NAD+ redox by 24 h of culture, measures of cellular energy state, [ATP], [ADP], [ATP]/[ADP] ratio, ([ATP] + [ADP] + [AMP]), [ATP]/([ATP] + [ADP] + [AMP]) and energy charge remained essentially constant until 72 h, despite periods of increased energy turnover. At 24 h there was a burst of glycogenolysis, and glycolysis indicated by increased lactate production, which coincided with formation of syncytium. Subsequently, there was no resynthesis nor further breakdown of glycogen. At 48 h, oxygen consumption temporarily increased substantially, without increased glycolysis, during functional differentiation of the syncytiotrophoblast. Glucose uptake was constant and largely from the basal (in vivo fetal facing) side. Lactate output into the basal fetal medium was twice as fast as that into the microvillous (maternal) medium, and oxygen uptake was also asymmetrical. The results show that before and after differentiation substantial relatively constant aerobic glycolysis occurs, but that during increased energy demand cytotrophoblast depends on both glycolytic and aerobic energy production whereas syncytiotrophoblast relies on aerobic metabolism.

Culture of syncytiotrophoblast for the study of human placental transfer. Part I: Isolation and purification of cytotrophoblast.

Criteria for a successful model for the study of trans-syncytiotrophoblast transfer include isolating substantially pure trophoblast cells from placental villous tissue, and obtaining from them phenotypical villous syncytial syncytiotrophoblast during culture. For studies involving the basal membrane, including overall transfer, basal uptake and output, and controls acting at the basal membrane, a two-sided model is required with a separate compartment of culture medium in contact with the basal cell surface. All current methods of isolating cytotrophoblast, the precursor of syncytiotrophoblast, derive from the original tissue trypsinization method of Thiede (1960), which produces cultures of villous cytotrophoblast cells contaminated with other placental cell types. Lessons learned from successful and unsuccessful development of the model over 35 years are outlined, and recently established methods for purifying the isolated mixed cells discussed. These include sedimentation and centrifugation methods, immunological and receptor binding methods, and more selective release of trophoblast cells from tissue. Immuno flow cytometric cell sorting methods are potentially capable of isolating subpopulations of various phenotypical trophoblast types. We conclude that satisfactory methods are now available for isolating and purifying cytotrophoblast from early or late gestation human placenta.

Culture of syncytiotrophoblast for the study of human placental transfer. Part II: Production, culture and use of syncytiotrophoblast.

The conditions necessary for producing syncytical syncytiotrophoblast are examined. Tissue disaggregation conditions, culture media composition, different extracellular matrices and the influence of placental gestational age are all assessed. The importance of evaluating the biochemical and functional differentiational state of the cells is also stressed. Evidence is summarized that syncytiotrophoblast in culture is morphologically and ultrastructurally very similar to syncytiotrophoblast in vivo, and what is so far known biochemically is largely consistent with what is known in vivo. Studies published to date on microvillous membrane uptake and release and relationships with intracellular metabolism using syncytiotrophoblast in conventional culture are outlined from the point of view of the advantages and potential of this model. The present state of development of the two-sided model is assessed, mentioning factors to be considered such as the supporting membrane to be used, accounting for passive diffusion and paracellular leak components of transport and dealing with quantitative effects in kinetic studies of the presence of the supporting membrane. It is concluded that satisfactory methods are now in place for preparing pure villous syncytial syncytiotrophoblast in culture from cytotrophoblast derived from term (but not early) placentae, suitable for studying microvillous membrane transport and relationships with intracellular metabolism. Cytotrophoblast from early gestational age placenta may require different conditions to form true syncytiotrophoblast. A two-sided model for studies of overall transfer, basal transport and basal control mechanisms is now available and possibly with some development should be a good model for such investigations.

Two-sided culture of human placental trophoblast. Morphology, immunocytochemistry and permeability properties.

We describe the culture of human term placental trophoblast cells on cell-free amniotic membrane, with medium on both sides. Over the course of 2 days, the isolated cells, initially simple, mononucleated and probably cytotrophoblast, form a confluent layer of multinucleated syncytial cells with morphological and immunocytochemical properties of syncytiotrophoblast. This layer becomes polarized with respect to morphology, alkaline phosphatase distribution and hCG secretion. Contamination with amnion cells, and with other cell types that are present in placental tissue, was less than 1 per cent. A preliminary investigation of the permeability properties of the preparation showed that the trophoblast cell layer, rather than the amniotic membrane, was rate-limiting to transtrophoblast transfer, but that possible effects of the supporting membrane should be considered. The transtrophoblast transfer of D-glucose and the non-metabolisable analogue, 3-O-methyl-D-glucose (3OMG), had saturable and non-saturable/leak components in both directions, indicating that carrier-mediated processes were involved. The non-metabolisable amino acid 2-aminoisobutyrate (AIB) was both accumulated within the trophoblast cells, and transferred by saturable and non-saturable processes from the microvillous side, but no saturable accumulation or transfer was observed from the basal side, at the concentrations tried. The results suggest that this model may prove suitable for studies of transtrophoblast transfer.

Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short-term culture.

Trophoblastic cells, of at least 95 per cent purity by immunofluorescence and morphological criteria, were obtained from human term placenta by a simple trypsinisation method without the additional purification steps or complex culture conditions used by others. The differentiation of these cells was followed over four days in culture by fluorescence immunocytochemistry, by scanning and transmission electron microscopy and by light microscopy. The results support the idea that the isolated cells are cytotrophoblast and that these differentiate during this time into cells with characteristics of villous syncytiotrophoblast. This process involved first the formation of a multicellular layer of mononucleated cells, then the development of a syncytium of multinucleated cells and, not necessarily concurrently, functional differentiation. This may be a useful model for the study of syncytiotrophoblast function.

Non-protein bound zinc concentration in human plasma and amniotic fluid measured by ultrafiltration.

Using an ultrafiltration technique, apparent non-protein bound (NPB) zinc concentrations in plasma were found to be 2.2 +/- 0.2 (SEM) microgram Zn/l (10 observations) in normal males, 1.6 +/- 0.3 (10) micrograms/l in normal females and 1.2 +/- 0.2 (10) micrograms/l in pregnant mothers during their 16th week of gestation. These values are about 0.2% of the total plasma zinc concentrations, at least five-fold less than previous estimates. In amniotic fluid, the NPB-zinc concentration was 12.6 +/- 0.4 (10) micrograms/l, 5-10 times that of normal plasma, though the total zinc concentration (100 +/- 30 micrograms/l) was only one tenth that of plasma. When plasma or amniotic fluid samples were ultrafiltered without precaution against CO2 loss, their NPB-zinc concentrations increased, suggesting that pH changes alter zinc binding. The low concentration of NPB-zinc in plasma explains the low urinary excretion of zinc observed by others and would be expected to restrict the entry of zinc into cells.

Effects of portocaval anastomosis on behaviour and brain tryptophan metabolism.

The rat with portocaval anastomosis represents a convenient took for the investigation of (a) factors determining the availability of tryptophan to the brain and (b) the role, if any, that altered tryptophan metabolism may have in the development of hepatic encephalopathy. It was found that increases in brain tryptophan following anastomosis paralleled plasma free tryptophan rather than plasma total tryptophan regardless of whether or not correction for inhibition from amino acids competing with tryptophan for uptake into brain was applied. Nevertheless, while plasma free tryptophan exerts a major influence on brain tryptophan in both sham-operated and anastomosed rats, in the latter group brain tryptophan is raised further by some other mechanism. The anastomosed rat was also found to be behaviourally abnormal in a number of test situations. Thus, they were hypoactive during chronic exposure to an open-field and were less responsive to electric shock. Following the administration of tryptophan, sham-operated rats were also less active in an open-field and less responsive to electric shock when compared with saline-treated rats. Thus, anastomosed rats have behavioural abnormalities for which altered tryptophan metabolism might, to some extent, be responsible.

Effects of chronic experimental liver dysfunction and L-tryptophan on behaviour in the rat.

Rats with chronic experimental portocaval anastomosis were hypoactive as indicated by diminished activity in the home cage, during habituation in red light to an observation box and during exposure in white light to an open-field. Food intake and responsiveness to electric shock were also decreased. However, there was an abnormally high frequency of social activity when anastomosed rats were paired together after having been caged singly for 3 weeks. Also, sham-operated rats interacted more with anastomosed rats than they did with other sham-operated animals. Anastomosis also raised brain concentrations of tryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. Administration of tryptophan to sham-operated rats increased shock threshold and decreased ambulation in an open-field. Thus, while anastomosed rats are not comatose they do have considerable behavioural abnormalities for which brain tryptophan changes may be in part responsible.

Brain 5-hydroxytryptamine metabolism after portocaval anastomosis: relationship with ambulation.

Chronic portocaval anastomosis (PCA) increased tryptophan and 5-hydroxyindoles in rat brain regions. PCA rats ambulated less than sham operated animals but showed positive correlations between ambulation and indices of brain 5-hydroxytryptamine (5-HT) metabolism, i.e., plasma free tryptophan, plasma non-esterified fatty acid, tryptophan in all brain regions, and 5-HT or 5-hydroxyindoleacetic acid in some regions. Results suggest a positive and non-causal association between motor activity and 5-HT metabolism in the PCA rats reflecting parallel responses of both variables to environmental stimulation. This may be superimposed on a normal inverse and causal relationship between motor activity and 5-HT metabolism.

Human fetal endothelial cells acquire zinc(II) from both the protein bound and nonprotein bound pools in serum.

To help determine physiologically important routes by which zinc (Zn) is acquired by human fetal vascular endothelium, the authors incubated cultured umbilical vein endothelial cells with 65Zn(II)-tracer labeled human fetal whole serum, ultrafiltrate (containing low molecular mass serum zinc complexes), and dialyzed serum (containing protein-bound zinc). Zinc from whole serum and from both serum fractions entered a rapidly labeled cellular compartment, removable by edetic acid (EDTA), representing Zn bound to the outside cell surface, and accumulatively, an EDTA-resistant compartment-probably largely internalized Zn. Entry of Zn into the EDTA-resistant pool from both serum fractions was strongly temperature-dependent, and was not via the EDTA-sensitive pool. Entry from the ultrafiltrate was resolvable into high affinity saturable, and non- (or hardly-) saturable components. Transfer from the dialyzed serum fraction was not significantly saturable, but only partially accounted for by nonspecific pinocytosis. Thus, Zn is obtained by fetal vascular endothelium partly from low molecular mass serum species, probably through at least one carrier-mediated membrane transport system; but also from Zn complexed with serum protein, via at least one metabolism-related route.

Restriction of hepatic gluconeogenesis and ureogenesis from threonine when at low concentrations.

Experiments were carried out with the isolated perfused liver of the overnight-starved rat to study the control of the conversion of the essential amino acid threonine to glucose and urea from the point of view of its conservation when in short supply. The relationships between the concentration of added L-threonine and the rate of glucose and urea production showed that both pathways have considerable capacity and were saturated at a high (15 mM) concentration of threonine. However, these concentration-rate relationships were sigmoidal, so that at low concentrations the rates of conversion were disproportionately low. Thus at physiologic levels of threonine, no measurable stimulation of glucose or urea output was observed. Hepatic uptake of threonine was similarly disproportionately reduced at near-physiologic levels. Glucagon stimulated glucose and urea outputs in parallel fashion and stimulated the uptake and inward membrane transport of threonine at both saturating and low concentrations. This and the changes in intracellular and extracellular concentrations of threonine indicate the transport is rate limiting for both pathways. If this is so, the apparent restrictive property probably resides at the plasma membrane. Since the liver is the end point of threonine metabolism, this property would effectively limit the utilization of threonine when in short supply.

Two major pathways of zinc(II) acquisition by human placental syncytiotrophoblast.

Uptake of zinc into placental villous syncytiotrophoblast is the first step in its transfer from mother to fetus. To help characterise physiologically significant pathways of zinc accumulation by these cells, we incubated cultured layers of syncytiotrophoblast cells derived from human near-term placental tissue with serum ultrafiltrate (containing the zinc complexed with low molecular mass serum constituents), dialysed serum (containing the zinc bound to the serum proteins) and whole serum, each of whose endogenous zinc was tracer-labelled with 65Zn(II). Zinc label from both fractions of serum readily entered a rapidly labelled EDTA-sensitive cellular compartment, probably representing zinc bound to the outside cell surface and in accumulative fashion, an EDTA-resistant compartment, probably consisting largely of internalised cellular zinc. Movement of zinc into the EDTA-resistant pool was strongly temperature-dependent and did not occur via the EDTA-sensitive pool from either serum source. Transfer of zinc from the low molecular mass serum fraction into the EDTA-resistant pool was saturable, the concentration giving half-maximal rate being 1.2 mumol/l nonprotein-bound zinc. No nonsaturable component was detected. Zinc from the serum protein-bound fraction entered by a saturable component, already saturated at physiological total protein-bound zinc concentration, and by an apparently nonsaturable component, not appreciably accounted for by nonspecific fluid-phase endocytosis. The results show that zinc is acquired by placental syncytiotrophoblast from the low molecular mass serum zinc pool probably by a carrier-mediated process, and at least as importantly, from the zinc bound to serum protein, possibly by an endocytic mechanism.

Condition and performance of the perfused human placental cotyledon.

The perfused human placental cotyledon was examined with respect to its viability, metabolic state, and performance. During the ischemic period before the start of perfusion, tissue adenosine triphosphate concentration and other measures of energy state fell rapidly to about half of the estimated in vivo value. During the subsequent perfusion, energy levels remained relatively stable but did not recover appreciably toward in vivo values. A very low transplacental leakage of inulin and a small cellular potassium loss indicate relative intactness of membrane function, but there were differences from the in vivo state in levels and balance of metabolic regulators adenosine triphosphate, adenosine monophosphate, and adenosine triphosphate/adenosine monophosphate ratio, and a more reduced cytoplasmic reduced nicotinamide adenine dinucleotide/ionized nicotinamide adenine dinucleotide couple. However, rates of oxygen and glucose consumption and lactate production and the maintenance of physiologic upward maternal-to-fetal concentration gradients of amino acids lead us to conclude that despite differences in energy and metabolic states the perfused cotyledon remains substantially intact and functions in certain respects comparably with the in vivo state.

An enzymatic fluorometric assay for L-lysine in plasma and tissue.

A simple, specific, and reliable method has been developed for the determination of L-lysine in blood plasma and tissue. The L-lysine in the sample is decarboxylated enzymatically, and fluorescamine is added to a pentan-1-ol extract of the cadaverine formed. This produces a stable product which is measured fluorometrically.

Foetal glutamate as a possible precursor of placental glutamine in the guinea pig.

The role of foetal glutamate as a source of placental glutamine was investigated in the near-term pregnant guinea-pig placenta perfused in situ through the umbilical vessels. With normal foetal amino acid concentrations there was a significant two-way exchange of glutamate between the placenta and foetal perfusate, but a net release of the amino acid from the placenta. Radioactively labelled glutamate carbon entering the placenta by this exchange was freely incorporated into intracellular glutamine, but only 1.5% of it was found in glutamine transported out into the foetal circulation. In the guinea pig, therefore, foetal glutamate does not appear to be a precursor of glutamine released from the placenta on the foetal side.