Nelvio Cester

A biochemical-morphological study on microvillus plasma membrane development

The microvillus plasma membrane of the human placental syncytiotrophoblast at term has been extensively studied, while little is known about the characteristics of its development. The aim of the present work was to compare functional and structural properties of this membrane at early and term gestational age. Ten normal term placentas (40 weeks) and ten placentas at 10 weeks of gestational age were studied. The Na+/K+-ATPase activity is significantly decreased in the syncytiotrophoblast plasma membrane obtained from term placentas as compared to the early ones, with significant variation of maximum velocity (Vmax). The microviscosity, evaluated by the P parameter of DPH and Sn parameters of 5- and 16-NS, is increased in the term placentas compared to the early placentas. This alteration is accompanied by an increased cholesterol to phospholipids ratio in term placentas, while there is a decreased unsaturated to saturated fatty acid ratio. As follows from morphological studies, an increased mean diameter in the E face was observed in the term placenta with respect to the early placenta. The distribution factor DF, which indicates the particle aggregation state, decreased in the E face in the term placenta as compared to the early one. The present biochemical morphological study shows that a deep modification of the membrane is at the basis of the syncytiotrophoblast plasma membrane development.

Modifications in platelet membrane transport functions in insulin-dependent diabetes mellitus and in gestational diabetes

The pathogenesis of plasma membrane alterations present in diabetes mellitus is unclear. To add new insights to the question, platelet membrane properties were evaluated in 16 women presenting impaired glucose tolerance at the 28-29th week of gestation (GDM) and in 8 women with insulin-dependent diabetes mellitus (IDDM). 15 healthy pregnant women (HPW) and 21 healthy non-pregnant (HNPW) women were the control group for GDM and IDDM, respectively. Pregnancy (HPW vs. HNPW) provoked an increase in Ca(2+)-ATPase activity and a decrease in membrane fluidity; in contrast, Na+/K(+)-ATPase, intracellular free Ca2+ concentrations, membrane cholesterol and phospholipid content did not vary. Both GDM and IDDM showed lower Na+/K(+)-ATPase activity and higher Ca2+ concentration, compared to HPW and HNPW, respectively, whereas Ca(2+)-ATPase activity was higher only in IDDM; furthermore, membrane fluidity was lower in GDM and higher in IDDM. Finally, GDM showed higher membrane cholesterol content. Both GDM and IDDM showed a very good metabolic control so that variations reported cannot be due to hyperglycemia; it is tempting to suggest that membrane variations are present before the clinical metabolic alteration. Furthermore, both GDM and IDDM were on insulin therapy, therefore: (i) insulin may be the pathogenetic factor of higher intracellular free Ca2+ concentrations and lower Na+/K(+)-ATPase activity since they both varied accordingly in GDM and IDDM, but not of (ii) changes in Ca(2+)-ATPase, membrane fluidity and cholesterol content which did not vary accordingly in GDM and IDDM.

Pregnancy Induced Hypertension: Observations on Chemical-Physical Properties of Syncytiotrophoblast Plasma Membranes from Human Placenta

The Pregnancy Induced Hypertension (PIH) was associated to alterations of ion transport, which may be due to abnormalities of membrane composition and function.We studied the syncytiotrophoblast plasma membranes of PIH mothers compared with normal ones.We observed a decreased microviscosity and an alteration of cholesterol to phospholipid ratio, together with an increased percentage of unsaturated fatty acid respect to the controls.We therefore hypothesized an involvment of the chemical-physical properties of the syncytiotrophoblast plasma membranes in the modified functional properties observed in the PIH.

Human hypertensive placenta contains an increased amount of Na,K-ATPase with higher affinity for cardiac glycosides.

Placentas of women suffering from pregnancy‐induced hypertension (PIH) were found to contain a greater amount of Na,K‐ATPase molecules, estimated from anthroyl ouabain binding, than normotensive individuals. Both the microsomal fraction of placental cells and purified Na,K‐ATPase showed an increased affinity for the specific inhibitor ouabain which, in the case of the microsomes, bound with a dissociation constant of 0.9 nM as compared with 3.4 nM in the controls. Likewise, the dissociation constant of the ouabain complex with purified Na,K‐ATPase was about 3.5 times lower in the hypertensive patients. The differences are apparently caused by a different microenvironment of the ouabain‐binding site, as reflected in the quantum yield of bound anthroyl ouabain. If an endogenous digitalis‐like factor is present in the body fluids to regulate Na,K‐ATPase activity, the present results render its role quite plausible.

Modifications induced by insulin-dependent diabetes mellitus on human placental Na+/K+-adenosine triphosphatase

The causes of the reduced activity of Na+/K+-adenosine triphosphatase (ATPase) in human diabetes are still the object of controversy. The aim of this work was to investigate the mechanisms of inhibition by means of the study of the Na+/K+-ATPase purified from human placenta. We purified Na+/K+-ATPase from term placentas of six healthy women and six age-matched women with insulin-dependent diabetes mellitus (IDDM) in good metabolic control. The enzymatic activity was reduced in both the microsomal fraction and the purified Na+/K+-ATPase obtained from diabetic women, whereas no difference was found in the number of active molecules determined by anthroyl ouabain binding. The Na+/K+-ATPase purified from women with IDDM did not show any modification in the ouabain affinity or changes in the physicochemical structure of the ouabain binding site investigated by dynamic fluorescence or alterations in lateral diffusion. The activation energy of the enzyme was increased, whereas the tryptophan accessibility of the enzyme was lower in women with IDDM. The fluidity of the lipid anulus of the enzyme was higher in women with IDDM than in control women, as suggested by fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene. The adenosine triphosphate-binding site, investigated by anisotropy decay studies of the fluorescent probe pyrene isothiocyanate, was modified in women with IDDM. It appears that the Na+/K+-ATPase of human placenta is altered in its disposition in IDDM.

Modifications induced by gestational diabetes mellitus on cellular membrane properties

Alterations in erythrocyte plasma membrane properties (enzymatic activities and membrane fluidity) have been observed in patients affected by insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). In order to verify whether these alterations are present also in gestational diabetes mellitus (GDM) we studied the plasma membranes obtained from two different cellular types (erythrocyte from both mother and cord blood and placenta syncytiothrophoblast cell) of 16 healthy pregnant women and 15 women affected by GDM. The following determinations were performed on the membrane preparations: Na+/K(+)-ATPase activity, acetyl-cholinesterase (AchE) activity, membrane fluidity and cholesterol:phospholipid ratio. We observed a reduction of both enzymatic activities and a decrease of membrane fluidity in maternal and cord blood erythrocytes and in syncytiotrophoblast plasma membranes in GDM pregnant women in comparison with controls. The cholesterol to phospholipid ratio was significantly lower in the erythrocyte membranes of women affected by GDM than in normal pregnant women, while it was increased in the cord blood erythrocyte membranes and in placental membranes in GDM in comparison with controls. The present study found, in GDM patients, a membrane alteration similar to the abnormality reported in IDDM and NIDDM (i.e. decreased Na+/K(+)-ATPase activity), while opposite modifications were observed with regard to other membrane activities and properties. The different membrane alterations observed in GDM with respect to IDDM and NIDDM might be linked to the different degree of metabolic control, on the contrary the reduced Na+/K(+)-ATPase activity might be a primary event in the pathogenesis of diabetes mellitus per se and might constitute a signal of high risk of developing the disease later in the women affected by GDM during pregnancy.

Plasma lipids and physicochemical properties of the erythrocyte plasma membrane throughout pregnancy

Background.  The physiopathological relevance of plasma lipid concentrations is supported by the observation that they might affect the physicochemical properties of the plasma membrane of circulating cells and might be crucial in the pathological conditions complicating pregnancy.

Calcium and sodium transport in gestational hypertension

The enzymatic activities of Na+/K+ ATPase and Ca2+ ATPase were determined on erythrocyte membranes from 9 normotensive and 9 gestational hypertensive pregnant women near term. A reduction in the activity of the Na+/K+ ATPase and a relative increase in the activity of the Ca2+ ATPase were found in the hypertensive patients, possibly due to a conformational alteration of erythrocyte membranes. This observation supports the possible role of the transmembrane cation transport in the pathogenesis of gestational hypertension.

Effect of Ca2+ on Structure and Fluidity of Microvillus Membranes of Human Placenta

This investigation shows the effect of a Ca2+ addition on the structural and physiochemical properties of microvillus plasma membranes obtained from human placenta. Ca2+ addition induces an increase in microviscosity, as shown by the increase of order parameter and rotational correlation time of 5- and 16-doxylsterate derivatives and by the increase of fluorescence polarization of diphenylhexatriene. All the effects were obtained in a wide temperature range. The morphometric analysis of the ultrastructural images shows that the vesicle profiles of syncytiotrophoblast membranes decrease both area and form factor (FF) in the presence of Ca2+ with respect to the controls. The freeze-fracture results also show that Ca2+ induces an enhanced tendency to IMP clusterization. The Ca2+-induced changes were observed in both E and P faces. Our results underline the important role of Ca2+ in the cell membrane structure per se and in modulating interactions between cytoplasmic and extracellular microenvironments. The results of morphometric analysis of the ultrastructural images agree with biochemical data showing an increased stability induced by calcium on plasma membranes.

Growth retardation and discordant twin pregnancy. An immunomorphological and biochemical characterisation of the human umbilical cord

Growth retardation and low birth weight represent an important factor associated with the high risk of mortality and morbidity, particularly in twin pregnancy, since twins are frequently characterised by fetal growth retardation and sometimes by a discordant growth between the twins. The present work sets out to elucidate the role of growth discordance between twins in the behaviour of human umbilical vein endothelial cells; biochemical, ultrastructural and immunohistochemical data obtained from a discordant twin pregnancy are discussed. Endothelial cells were obtained from umbilical cord of 5 singleton pregnancies and from 5 dichorionic twin pregnancies, among which was one discordant twin pregnancy. Membrane fluidity was assayed using a fluorescent probe and each sample was immunohistochemically processed employing specific monoclonal antibodies. An increased fluidity was observed in endothelial cells from the smaller twin as compared with the larger one. As concerns electron microscopy, the features of endothelial cells from the smaller twin were similar to those of twins with similar growth, while endothelial cells from the larger one were more similar to those from singleton pregnancies. Our findings confirm the presence of the feto‐fetal transfusion as a pathogenetic mechanism involved in twin growth discordancy.