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An altered interaction between circulating LDL and endothelial cells might be at the basis of the increased prevalence of atherosclerosis in diabetes mellitus. The aim of the present work was to investigate the effect of a short incubation period with LDL from Type 1 diabetic patients in good metabolic control on endothelial cells derived from human aorta (HAEC). Cultured HAEC were incubated for 3 h with culture medium alone (control HAEC), with native LDL from healthy subjects (control LDL), or with native LDL from Type 1 diabetic patients (Type 1 LDL). After the incubation the following parameters were evaluated: endothelial cell nitric oxide synthase (NOS) activity, nitric oxide (NO) and peroxynitrite production, Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities, intracellular Ca(2+) concentration and fluidity of the superficial part of the plasma membrane studied by 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Moreover, we studied the cellular activation, evaluated by the fluid phase endocytosis of TMA-DPH, and the microetherogeneity of the membrane surface, evaluated by dynamic fluorescence. HAEC incubated with control LDL showed compared with control HAEC: increased anisotropy and exponential lifetime of TMA-DPH, and enhanced TMA-DPH internalization. HAEC incubated with Type 1 LDL showed compared with both control HAEC and HAEC incubated with control LDL: (i) increased Na(+)/K(+)-ATPase and Ca(2+)-ATPase activities, and intracellular Ca(2+) concentration; (ii) increased NOS activity, NO and peroxynitrite production; (iii) increased anisotropy of TMA-DPH; (iv) enhanced internalization of the probe. The exponential lifetime and the width of distribution of TMA-DPH were significantly increased by Type 1 LDL only in comparison with control HAEC. The results suggest that a short-term interaction with LDL from Type 1 diabetic patients causes alterations of the plasma membrane surface and of cellular functions in endothelial cells in a possibly atherogenic way.

Background 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. Design 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. Results 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. Conclusion 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.

Decreased levels of nitric oxide (NO) may contribute to impaired endothelium-dependent vasodilatation in essential hypertension. Moreover, in hypertension, major platelets aggregation and endothelial adhesion, and increased atherogenetic risks are also present. Nitric oxide produced by platelet NO synthase, which is similar to endothelial NO synthase, inhibits platelets aggregation by increasing cytoplasmic cyclic GMP levels and contributes in a major way to the antithrombogenic properties of endothelium. The aim of this study was to investigate platelet NO production and cytosolic Ca2+ levels in patients with essential hypertension and in healthy subjects. We studied NO production in 36 subjects (21 patients had essential hypertension and 15 subjects were normotensive); NO synthase activity was evaluated by measuring nitrite levels by the Griess reaction in the supernatant of sonicated platelets. Cytosolic Ca2+ levels were measured in intact platelets using the fluorescent probe Fura 2-AM. Nitric oxide levels in platelets were found higher in normotensive than in hypertensive patients (P < .0001). Nitric oxide levels in hypertensive women were significantly higher than in hypertensive men (P < .001). Hypertensive women and men had lower levels of nitrite than normotensive women and men (P < .001 and P < .002, respectively). Platelet cytosolic Ca2+ levels were higher in hypertensive patients than in normotensive subjects (P < .001). An inverse correlation was found between platelet cytosolic Ca2+ and NO levels (r = 0.74, P < .002). These data confirm the link between hypertension and altered platelets function and suggest a role for NO in cardiovascular events. Moreover, the higher levels of nitric oxide in child-bearing age women than in men further support the protective effect of estrogens on cardiovascular diseases.

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.