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In order to investigate the molecular mechanisms of the inhibition of Na+/K+-ATPase in Gestational Hypertension (GH), we incubated Na+/K+-ATPase purified from human placenta of 6 healthy normotensive women with plasma from 6 GH women and 6 healthy controls. We determined the enzyme activity by the method of Esman, and the anthroyl-ouabain-binding capacity, dissociation constant (Kd) and average lifetime values (tau) by the static and dynamic fluorescence of anthroyl-ouabain. The lipid annulus of the enzyme was studied by static and dynamic fluorescence of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5- hexatriene (TMA-DPH). The addition of total and protein-free GH plasma to normal Na+/K+-ATPase significantly inhibited the enzymatic activity even at the lowest concentration studied (1:100), as well as the ouabain-binding capacity, Kd and tau. GH plasma significantly decreased the fluorescence polarization and lifetime values of TMA-DPH. These observations indicate that the inhibition caused by GH plasma on Na+/K+-ATPase might be due to a reduction of the number of active molecules or a modification of the ouabain-binding site suggesting the existence of digitalis-like factor. A link between the modification of the lipid moiety of the enzyme and the Na+/K+-ATPase inhibition might be hypothesized.

A decrease of Na+/K(+)-ATPase activity has been reported in syncytiotrophoblast plasma membrane (SPM) obtained from pregnancy induced hypertensive (PIH) women. The aim of the present work was to verify if the reported modifications in activity are due to a decreased number of enzymatic molecules or to a conformational change of the enzyme itself. Morphological studies were performed in order to better understand the relations between the enzymatic protein and the lipid bilayer. Kinetic studies were also performed. SPM obtained from PIH showed: i) an increased affinity of Na+/K(+)-ATPase for ouabain binding, ii) a significant change in the maximum velocity of the enzyme, iii) a higher distribution factor (DF) of intramembrane particles (IMPs) in the exoplasmic face of the membrane, iv) a decreased mean diameter of IMPs both in the protoplasmic and exoplasmic faces, v) a decreased number of IMPs in the exoplasmic face. In conclusion, a conformational modification seems to be at the basis of the decreased Na+/K(+)-ATPase activity during PIH as suggested by binding, ultrastructural and kinetic data herein reported.

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 an in vitro oxidative stress as a tool to verify the predisposition to the in 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.(ABSTRACT TRUNCATED AT 250 WORDS)