Pregnancy induced hypertension: a role for peroxidation in microvillus plasma membranes

It has been recently hypothesized that in PIH a placental oxidant-antioxidant imbalance might cause the release of lipoperoxidation products into the circulation, with subsequent damage of endothelial cell membranes. In this hypothesis the endothelial cell and further increase in circulating lipoperoxide levels, which are by themselves able to induce smooth muscle constriction and increased pressor responsiveness to angiotensin II. In order to investigate this issue, we studied the basal content of lipid peroxides in terms of malondialdehyde (MDA) in the syncytiotrophoblast plasma membranes (SPM) from PIH women. Moreover, we investigated the susceptibility to peroxidation of SPM using anin vitro oxidative stress as a tool to verify the predisposition to thein vivo development of peroxidation products. The fatty acid composition of the membranes was also analyzed. Microvillus membrane lipoperoxide concentrations were significantly increased in PIH women (62.8±7.6 ng MDA/mg prot) compared with healthy pregnant subjects (37.6±4.8 ng MDA/mg prot; p<0.01).The formation of TBARS under the action of phenylhydrazine was significantly greater in PIH women (90.3±7.4 mmol MDA/mol cholesterol) than in normal pregnant subjects (68.6±6.4 mmol MDA/mol cholesterol; p<0.01). In PIH microvillus membrane we also observed a significant increase of the content of polyunsaturated arachidonic acid.The increased susceptibility to oxidative stress of SPMs from PIH women might be due either to reduced antioxidant systems or to an abnormality of the lipid composition of the membrane. The present work also demonstrated in PIH a reduction in the SPM content of saturated fatty acids with an increase in polyunsaturated fatty acids, which are the major substrate for peroxidation. On the other hand, the higher lipoperoxidation may be due to the observed increased susceptibility to peroxidative stress, to a primary reduction in placental perfusion with tissue hypoxia or to both factors, which can potentiate each other.     

Changes in membrane fluidity and Na+/K(+)-ATPase activity during human trophoblast cell culture.

The human placenta plays an essential role in embryo development, in particular regulating the transport of ions, nutrients and immunoglobulins from the maternal to the fetal circulation. Trophoblast organization into a syncytial layer involves structural and functional steps that may be monitored and elucidated by in vitro studies. The structural stages by which the syncytial trophoblast is formed are not yet understood. In order to clarify the mechanism of trophoblast development, we studied the morphological characteristics of the syncytial trophoblast formation in culture and the functional changes (transport properties and membrane microviscosity) accompanying the structural modifications. By using both 5-nitroxystearate and 16-nitroxystearate as spin labels, we observed an initial increase in membrane order over 0-24 h of culture, which can be associated with two events: recovery of cell membranes from trypsin and initial aggregation of cytotrophoblasts. The similar behaviour of the order parameters determined with both probes indicates that membrane order changes both inside and in the outer part of the lipid bilayer. The subsequent decrease in membrane order observed at 36-48 h might be related to the process of cellular fusion. The increase in sodium/potassium pump activity in the first 24 h of culture might be an expression of cell recovery following trypsin treatment. The subsequent decrease might represent an adaptive mechanism by which metabolic energy is mainly used for morphogenetic changes.     

Sodium metabolism in offspring of hypertensive parents.

Intracellular sodium concentration and Na+/K(+)-ATPase activity were studied in erythrocytes obtained from members of 14 families with one hypertensive parent and from age-matched control subjects, as part of a study on the genetic and environmental determinants of essential hypertension. We found reduced Na+/K(+)-ATPase activity, increased intracellular Na+ concentration, and reduced urinary Na+ excretion in hypertensive patients as compared with the control subjects. In the offspring of hypertensive parents an increase in intracellular Na+ concentration and a decrease in Na+/K(+)-ATPase activity were observed, with a significant correlation relating such parameters. Normotensive spouses did not differ from the normotensive control adults in any of the parameters studied, suggesting no influence of shared family environment in our family group. These data suggest that there is a strong genetic influence contributing to familiar alterations in cation transport, although long-term studies are needed to evaluate the influence of environmental determinants.     

Modifications induced by general anesthetics on Na+/K+ ATPase obtained from human placenta

Previously it was demonstrated that thiopental in vivo anesthesia didn't affect the Na+/K(+)-ATPase activity of syncythiotrophoblast plasma membrane, while affecting other enzymatic activity. The aim of the present work was to investigate if this lack of effect of thiopental on the Na+/K+ ATPase activity might be due to its specificity of action on definite membrane proteins or if the binding sites of the anesthetic to this enzyme might be masked within the membrane. Temperature dependence of the Na+/K(+)-ATPase activity and of a spin label paramagnetic maleimide derivative (MSL,2,2,6,6-tetramethylpiperidin-1-oxyl-4-maleimide), which shows a selective binding to the reduced sulfhydryl groups of proteins were investigated. This report shows that a Na+/K(+)-ATPase membranous preparation obtained from placental tissue is strongly inhibited by thiopental.     

Spermatogenesis in mice is not affected by histone H1.1 deficiency

The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S phase of the cell cycle. In adult mice the histone gene H1.1 is expressed mainly in thymus, spleen, and testis. The single-copy gene coding for the H1.1 protein was eliminated by homologous recombination in mouse embryonic stem cells. Mice homozygous for the deficient H1.1 gene developed normally until the adult stage without H1.1 mRNA and H1.1 protein. No anatomic abnormalities could be detected. In addition, mice lacking the H1.1 gene were fertile and they showed normal spermatogenesis and testicular morphology.     

Modifications induced by plasma of gestational hypertensive women on the Na+/K+-ATPase obtained from human placenta

Pyrimidines and purine (deoxy)nucleotides are the building blocks of DNA and RNA. Nucleoside diphosphate sugars, e.g. UDP-glucose, are the reactive intermediates in the synthesis of nearly all glycosidic bonds between sugars.In mammals the requirement for pyrimidines is met by UMP de novo synthesis and, to a greater or lesser extent, by salvage of free nucleosides. The exceptional compartmentation of the de novo synthesis with respect to mitochondrially-bound dihydroorotate dehydrogenase ('DHOdehase' or 'DHODH', EC 1.3.99.11) is one focus of the present work. DHODH activity was determined by the dihydroorotate-dependent oxygen consumption or by the UV absorption of the product orotate with mitochondria isolated from rodent and porcine tissues. For comparison, the cytochrome c and choline-dependent oxygen consumption of mitochondria from different tissues was measured. The highest specific activity of the rat DHODH was found in liver (2.3 × 10-3 µmol/min × mg protein) > kidney > heart. The application of known enzyme inhibitors Brequinar Sodium and Leflunomide for DHODH and sodium cyanide for cytochrome c oxidase verified the specificity of the activity tests used. The relation of DHODH activity versus that of cytochrome c oxidase revealed the lowest ratios in heart mitochondria and the highest in liver mitochondria. Since disorders in the mitochondrial energy metabolism could entail severe impairment of pyrimidine biosynthesis via respiratory-chain coupled DHODH, it is suggested to include improvement of pyrimidine nucleotide status in therapy protocols. (Mol Cell Biochem 174: 125–129, 1997)     

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.     

Effect of HMG-CoA reductase inhibitors on the erythrocyte membrane]

We studied 10 patients affected by primary hypercholesterolemia treated with placebo for 1 month and with simvastatin (20 mg die) for 6 months during a double-blind clinical trial. At 1-month intervals we determined the following parameters in the serum: total and HDL-cholesterol, triglycerides, apolipoprotein A1 and B. At the same time intervals, we also determined the cholesterol and phospholipid concentration, the Na+/K+ ATPase activity and the fluidity of the erythrocyte membranes. Our results demonstrated the following modifications in the erythrocyte membranes during simvastatin treatment: 1) an initial increase in the cholesterol concentration and in the cholesterol/phospholipid ratio, with a significant decrease only after 4 months; 2) a similar behaviour of membrane fluidity, with an initial decrease and an elevation after 4 months; 3) an increase in the Na+/K+ ATPase activity only after 4 months. We hypothesize that simvastatin not only inhibits the hepatic synthesis of cholesterol, but also modifies the cholesterol exchange between plasma and the erythrocyte membrane.     

Tyrosine phosphorylation in type-1 diabetes by immunogold detection: an in vitro human aortic endothelial cell (HAEC) study in the presence of diabetic low density lipoproteins (LDL)

An immunomorphometric study of tyrosine phosphorylation was performed by the immunogold technique on cultured human aortic endothelial cells (HAEC) with a view to demonstrating their impaired signal transduction status, induced in vitro by incubation with low-density lipoproteins from the plasma of Type-1 diabetic patients. The results seem to sustain the hypothesis that extranuclear bioenergetic derangement induced by low-density lipoproteins from Type-1 diabetic patients may be associated with an up-regulation of the nuclear energetic machinery aimed at maintaining intracellular metabolic equilibrium. Our data demonstrate that phosphorylated tyrosine is a useful marker to monitor this metabolic condition.