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Apoptosis in lymphoid cells can be induced in different ways depending on cell type and acquired signal. Biochemical modifications occur at an early phase of cell death while at late times the typical morphological features of apoptosis can be visualized. The aim of this study is to verify by multiparametric analyses the plasma membrane fluidity, the intracellular Ca2+concentration and the nitric oxide synthase (NOS) activity during cell death progression induced by DMSO treatment. The RPMI-8402 human pre-T lymphoblastoid cell line was induced to cell death by DMSO. Analyses rescued at early times of treatment prove a substantial modification of plasma membrane fluidity associated with an increase of intracellular Ca2+. Moreover, these modifications are associated with an up regulation of NOS activity. Our results are consistent with the hypothesis that programmed cell death can be induced by up regulation of the intracellular Ca2+associated with an increase of cell membrane fluidity. The apoptotic mechanisms seem to involve not only membrane damage and increased intracellular calcium levels but also production of nitric oxide.

The plasma membrane composition affects intracellular processes and the cellular susceptibility to free radical attack, which has been associated with the impairment of cellular functions occurring during senescence. The study of the modifications of the plasma membrane in centenarians might elucidate the biological mechanisms at the basis of longevity and successful aging. The work was performed in 190 subjects, divided into five groups according to the age range: (1) 21-40 years (n=25); (2) 41-60 years (n=30); (3) 61-80 years (n=30); (4) 81-99 years (n=50); and (5) centenarians (> or = 100 years) (n=55). The following determinations were performed on erythrocyte membranes: (i) the lipid peroxide level (Lp) evaluated as malondialdehyde content; (ii) susceptibility to in vitro oxidation evaluated as difference in the content of thiobarbituric acid-reactive substances before and after phenylhydrazine addition; (iii) unsaturated/saturated fatty acid ratio and individual polyunsaturated fatty acid composition measured by gas chromatography; and (iv) fluidity studied by means of the anisotropy of the probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Erythrocyte membranes from centenarians showed: (i) decreased basal lipid peroxide levels and reduced susceptibility to peroxidation in comparison with elderly subjects; (ii) increased unsaturated/saturated fatty acid ratio in comparison with every other age group; (iii) higher levels of eicosapentaenoic and docosahexaenoic acid and reduced content of linoleic and arachidonic acid in comparison with elderly subjects; and (iv) decreased anisotropy of TMA-DPH, i.e. higher fluidity compared with all the other age groups. In conclusion, the present work demonstrates that erythrocyte membranes from centenarians show some distinct features in comparison with elderly subjects that might act in a protective way against injuries.

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 (τ) 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 τ. 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; p0.01). 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; p0.01). 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)

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.

The molecular mechanisms involved in the platelet activation observed in hyperhomocysteinemia are not known. We aimed to discover if homocysteine concentrations are associated with abnormal platelet nitric oxide production in healthy and diabetic subjects. The study cohort included 28 patients with Type I (insulin-dependent) diabetes mellitus, 30 patients with Type II (non-insulin-dependent) diabetes mellitus, and 34 healthy subjects. Homocysteine plasma concentrations were measured by high-performance liquid chromatography. Platelet nitric oxide production was measured using a nitric oxide meter before and after a 3-h incubation with 100 μmol/l homocysteine. Stimulation experiments were done in vitro by the addition of α-thrombin (0.2 U/ml). Basal platelet nitric oxide production was lower in diabetic patients than in healthy subjects. Nitric oxide release was reduced by in vitro homocysteine incubation, being lower in platelets from diabetic patients than in platelets from control subjects. Thrombin increased nitric oxide synthesis in platelets from healthy subjects both in the presence and absence of homocysteine. In diabetic subjects thrombin increased nitric oxide release in the absence of homocysteine. But in the presence of homocysteine the response was reduced. An inverse relation was found between plasma homocysteine levels and basal platelet nitric oxide release in diabetic and healthy subjects. Homocysteine could exert its atherogenic action in healthy and diabetic subjects partly by inhibiting platelet nitric oxide production with the subsequent increased platelet activation and aggregation. [Diabetologia (2001) 44: 979–982]

Na+,K(+)-ATPase activity was evaluated in relation to membrane composition and molecular organization in erythrocyte membranes from obese patients by the amphyphylic molecule 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). Its possible relationship with fat distribution and hyperinsulinaemia was also investigated.Subjects were 10 obese men (OM), 12 women with subcutaneous obesity (FSO), 10 women with abdominal obesity (FAO) and 41 healthy lean subjects, 26 women (FC) and 15 men (MC). An oral glucose tolerance test was administered to all subjects to evaluate insulin secretion and glucose tolerance.Na+,K(+)-ATPase activity was increased in all obese patients. Values were higher in FSO and FAO than in FC (with FAO greater than FSO) and in OM than in MC. The erythrocyte membrane cholesterol-to-phospholipid ratio was increased in obese patients and was significantly different in FSO patients compared with FC. The erythrocyte membrane protein-to-phospholipid ratio was also increased in all obese subjects, reaching statistical significance only in FSO vs. FC. The liquid crystalline phase, as tested by Laurdan generalized polarization (GP), was decreased in obese patients, indicating the presence of greater molecular environmental order; all patients groups showed lower GP values than control subjects, but only FAO reached statistical significance compared with FC. There was no evident correlation between membrane Na+,K(+)-ATPase activity and insulin levels, nor did membrane composition and properties show any evident relationship with insulin levels.Both increased Na+,K(+)-ATPase activity and altered fluidity and lipid composition were observed in the erythrocyte membrane of all obese patients. These findings are in line with previous observations by our group and indicate that the changes in Na+,K(+)-ATPase activity observed in obese patients could be related to changes in plasma membrane organization and composition.

To investigate the molecular mechanisms of the inhibition of Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) in diabetes mellitus, we incubated Na+,K(+)-ATPase purified from human placenta of six healthy nondiabetic women with plasma from six insulin-dependent diabetic (IDDM) men and six healthy controls and with different concentrations of lysophosphatidylcholine (LPC). We determined the enzyme activity, 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-trimethylamino-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Moreover, we studied the lipid microenvironment surrounding the Na+,K(+)-ATPase purified from the placentas of six healthy women and six insulin-dependent diabetic women, determining the percent composition of phospholipids of the lipid annulus. The addition of total and protein-free IDDM plasma to normal Na+,K(+)-ATPase significantly inhibited the enzymatic activity even at the lowest concentration studied (1: 100), whereas the ouabain-binding capacity, Kd, and tau were not affected by IDDM plasma. The fluorescence polarization and lifetime values of TMA-DPH were significantly decreased by diabetic plasma. The incubation of Na+,K(+)-ATPase with LPC caused an inhibition of the enzymatic activity without modifications of the anthroyl ouabain-binding capacity and dissociation constant. The fluorescence polarization and lifetime values of TMA-DPH were significantly decreased by 5 mumol/L LPC. The study of the phospholipids surrounding Na+,K(+)-ATPase demonstrated a significant increase in the percent LPC content in IDDM patients compared with controls together with a concomitant decrease in phosphatidylcholine. These observations indicate that the inhibition caused by diabetic plasma on Na+,K(+)-ATPase is not dependent on a modification of the ouabain-binding site and that it seems to mimic the effect of LPC addition. A link between modification of the lipid moiety of the enzyme and Na+,K(+)-ATPase inhibition might be hypothesized.

Nitric oxide (NO) produced by platelet nitric oxide synthase (NOS) inhibits platelet activation by increased cytoplasmic cGMP levels. The aim of this study was to investigate platelet NOS activity in insulin-dependent (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM), which are characterized by enhanced platelet activation. HbA1 c levels, platelet NOS and platelet membrane Na+/K+ ATPase activity were determined in 19 IDDM patients, 21 NIDDM patients and 31 healthy control subjects. NOS activity was measured by a spectrophotometric method based on NO-dependent oxidation of oxyhaemoglobin to met-haemoglobin. Na+/K+ ATPase activity was measured by the method of Kitao and Hattori. Both NOS and Na+/K+ ATPase activity were significantly reduced in diabetic subjects compared with control subjects. NOS showed a significant negative relation with HbA1 c levels and a positive relation with Na+/K+ ATPase activity in diabetic patients. It is hypothesized that the decreased NOS activity might play a role in the pathogenesis of diabetic vascular complications. [Diabetologia (1998) 41: 101–104]

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.

The distance between the beta-subunits of Na+/K+-ATPase isolated from pig dark red kidney medulla was determined by Förster energy transfer. First, oligosaccharides of the beta-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B sulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSH were used as the donor and the acceptor, respectively, for Förster energy transfer studies to determine the localization of the beta-subunit in the native enzyme which is known to be formed as a tetramer (alphabeta)2. It was found that the beta-subunits in the functional enzyme complex in the membrane are not localized next to each other but are spatially separated. The distance between fluorophores covalently attached to the beta-subunits was found to be 5.1 nm. This conclusion was confirmed by measurements with another donor-acceptor pair CB-LY. The results also support the idea of a direct interaction of the beta-subunit with the extracellular part of the alpha-subunit. These interactions were modified in the presence of millimolar concentrations of magnesium ions. This indicates a crucial role of magnesium in extracellular interactions between the alpha and beta subunits.

Phase-modulation fluorescence lifetime measurements were used to study the single Trp residue of the Ca(2+)-binding protein S-100a both in the absence and in the presence of Ca2+ and/or Mg2+. Trp fluorescence decay for the protein was satisfactorily described by Lorentzian lifetime distributions centered around two components (approximately 4 ns and 0.5 ns). Lifetime values were unchanged by 2 mM Ca2+, but the fractional intensity associated with longer lifetime increased up to 75%. In the presence of Mg2+, the Ca2+ induced increase of the fractional intensity associated with longer lifetime was only 57%. For the protein in buffer, about the 85% of the recovered anisotropy was associated to a rotational correlation time of 6.7 ns. After the addition of Ca2+, this value was increased to 16.08 ns. In the presence of Mg2+, Ca+2 increased the rotational correlation time to 33.75 ns. Similar studies were performed with S-100a interacting with egg phosphatidylcholine vesicles (SUV). Our data suggest that the conformation of the protein may be influenced by structural features of the lipidic membrane. Moreover, data obtained in the presence of Mg2+ indicate some interaction between lipids and S-100, likely mediated by this ion.

Platelet functionality alterations have been correlated to the onset of hypertension in pregnancy and oral Mg++ supplementation has been clinically postulated to counteract such alterations. We, therefore tested the effect of 4 weeks oral Mg++ pyrrolidone carboxylate supplementation on platelet function. Forty-eight pregnant women were enrolled in the study at the beginning of the third trimester (30-32 weeks). Twenty women were preeclamptic, while 28 remained normotensive and served as controls. All the women received 360 mg/day magnesium pyrrolidone carboxylate for 4 weeks. DPH fluorescence, Na+/K(+)-ATPase and Ca(++)-ATPase activity, intracellular free Ca++ concentrations were determined prior and after the 4-weeks supplementation. Oral Mg++ supplementation significantly increased platelet DPH fluorescence in both normotensive and preeclamptic women. In normotensive pregnant women, it also significantly increased the activity of Na+/K(+)-ATPase, the activity of Ca(++)-ATPase and reduced the concentration of intraplatelet free Ca++. In hypertensive pregnant women, Mg supplementation increases Na+/K(+)-ATPase activity and decreases intracellular free Ca++; this, in turn, contributes to reducing the activity of Ca(++)-ATPase. Magnesium supplementation to preventing hypertension in pregnancy seems to have a consistent biochemical and clinical background.

Previous studies showed that anaesthesia with the barbiturate Thiopental induces an increase in membrane fluidity and a decrease in acetylcholinesterase activity in syncytiotrophoblast plasma membranes (SPM) obtained from placentas after Cesarean section. The aim of the present work was to compare the effect of a local anaesthetic (bupivacaine hydrochloride, trade name Marcaine) on SPM in vivo and to establish whether the anaesthetic is still present in the membrane after tissue preparation. The acetylcholinesterase activity was lower in Marcaine-anaesthetized SPM (27 +/- 3 against 39 +/- 6 in the control). The Marcaine action on the SPM can be ascribed to a competitive inhibition, similar to that reported for Thiopental. Fluorescence studies of the order parameter P showed it to be higher in SPM obtained from control (0.253 +/- 0.012) than in SPM obtained from Marcaine-exposed membranes (0.240 +/- 0.015). The local anaesthetic is still present in the SPM after their preparation (20.1 ng per mg membrane protein). It appears that the local anaesthetic exhibits an effect similar to that of the general anaesthetic, apparently due to binding to the membrane.

Several plasma membrane alterations have been described in diabetes mellitus. Data reported in gestational diabetes mellitus (GDM) suggested that these alterations might be present before the onset of overt metabolic derangement. On the basis of these data it is tempting to hypothesize that the reduction in the sodium pump activity might be due to a genetic factor acting at the membrane level before the onset of diabetes. In order to verify this hypothesis 11 insulin-dependent diabetic patients, 15 first degree relatives of the patients and 10 healthy subjects with a negative family history for diabetes mellitus were studied. Fluidity, Na+/K(+)-ATPase activity and membrane cholesterol content (C) were evaluated on plasma membranes obtained from red blood cells (RBCs). Na+/K(+)-ATPase activity was reduced with a contemporary increase in membrane fluidity in RBCs from IDDM patients in comparison to either relatives and controls. The same alterations were observed also in RBCs from the relatives in comparison to controls. We did not find any significant difference in the C content among the three groups. Data herein reported provide evidence that a reduction in the Na+/K(+)-ATPase activity is present in the plasma membrane of relatives of diabetic subjects. Furthermore, the present work suggests that the change in enzymatic activity might be related to modifications in membrane fluidity.

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.

Le gravidanze gemellari sono caratterizzate da riduzioni delle dimensioni del feto accompagnati dai corrispondenti riduzioni placentari, come precedentemente riportato. Anche modificazioni sono state evidenziate a carico degli altri annessi embrionali, mentre varie sono le problematiche non risolte. Pertanto noi abbiamo voluto valutare le caratteristiche morfofunzionali delle vene ombelcali cordonali di gemelli dicoriali confrontandoli con quelle di gravidanze normali a termine. Sono stati analizzati i cordoni ombelicali di gravidanze a termine e da una gravidanza gemellare dicoriale recisi dalla placenta subito dopo il parto. È stata utilizzata la microscopia elettronica a trasmissione (TEM) e a scansione (SEM). Per l'indagine immuno istochimica è stata impiegata la tecnica del complesso avidina-biotina perossidasi. Le cellule endoteliali sono state ottenute con il metodo di Jaffe e per gli studi di fluorescenza è stata utilizzata la sonda l-(4 trimetilaminofenil) 6 fenil-1,3,5 — esatriene (TMA-DPH).Nei cordoni ombelicali ottenuti da gravidanze normali a termine esse presentano una completa fase di differenziamento. Abbiamo infatti osservato: 1) elementi modicamente appiattiti, 2) protrusioni citoplasmatiche, 3) vescicole di pinocitosi e citoplasmatiche, 4) aggregati di lisomi, 5) corpi di Weibel e Palade, 6) nucleo lenticolare, 7) nucleo evidente con predominante eucromatina. La tonaca media appare ricca di fibrocellule muscolari lisce ricche di filamenti di tipo contrattile. Nei gemelli le celule endoteliali appaiono globose con aspetti di attività cellulare come anche confermato dagli studi biochimici. Le cellule muscolari sottostaminali risultano assai ricche di reticolo ergastoplasmatico e con fenotipo sintetico.In conclusione per quanto concerne il cordone ombelicale quanto da noi osservato istochimicamente ultrastrutturalmente biochimicamente pare sottolineare uno stato di minor maturità delle gravidanze gemellari rispetto ai controlli normali a termine.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.