Nitric oxide (NO) has been shown to inhibit platelet activation through an increase in cytoplasmic cGMP levels [1,2]. Recent evidence suggests that the cGMP increase induced by insulin in human platelets, which accounts for the antiaggregatory effect of the hormone, is mediated by NO [3]. NO production within the platelet is made possible by the presence of a constitutive isoform of the enzyme nitric oxide synthase (NOS) , which has been recently isolated from human platelets [4]. Platelet NOS has a distinct molecular weight (150 kDa) and its amino acid sequence has been deduced from its mRNA sequence [4].Low levels of intraplatelet cGMP have been described in insulin-dependent diabetic (IDDM) subjects and it has been hypothesized that this alteration might account for the platelet hyperreactivity during the disease [5]. However, no data are available at present in the literature directly concerning the study of platelet NOS in human diabetes, which is accompanied by an enhanced platelet activation.The aim of the present study was to investigate directly the platelet NOS activity in patients affected by IDDM and NIDDM and to look for possible relations with metabolic control. Moreover, we determined in the same subjects the platelet Na + /K + ATPase activity, as a relation has been hypothesized between this enzymatic activity and NO production in endothelial cells from diabetic rats [6]. Subjects and methodsThe study was performed on 19 IDDM outpatients (9 men, 10 women, age 34 ± 8 years, duration of disease 7 ± 5 years, fasting glycaemia = 9.5 ± 2.9 mmol/l, HbA 1c 9.0 ± 1.3 %, range 6.3--11.3 %), 21 NIDDM outpatients (10 men, 11 women, age Diabetologia (1998) Summary 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. HbA 1 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 HbA 1 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)

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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 Ca 2+concentration and the nitric oxide synthase (NOS) activity during cell death progression induced by DMSO treatment. The RPMI·8402 human pre-T Iymphoblastoid 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 Ca 2+. Moreover, these modifications arc 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 Ca 2+ 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.

Moderate increases in the plasma concentrations of homocysteine are associated with an increased incidence of arteriosclerosis and cardiovascular disease [1]. Homocysteine could exert adverse vascular effects by a number of mechanisms: production of reactive oxygen species caused by auto-oxidation, reduced endothelial generation of nitric oxide (NO) and/or enhancement of vascular smooth muscle cell proliferation [2]. Recent data have shown that flowmediated vasodilation is reduced in the presence of methionine-induced mild hyperhomocysteinemia and that this endothelial dysfunction is because of impaired NO activity without change of oxidative status [3]. Moreover, the exposure to homocysteine increases the interaction of leukocytes and endothelium through the expression of the endothelial cell adhesion molecules (P-selectin and ICAM-1) and the up regulation of CD18 expression in neutrophils [4]. A concomitant treatment with an NO donor reduces these homocysteine effects [4].Platelet-rich thrombus formation has been found in homocysteine-induced atherosclerotic lesions but Diabetologia (2001) Abstract Aims/hypothesis. 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. Methods. 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 mmol/l homocysteine. Stimulation experiments were done in vitro by the addition of a-thrombin (0.2 U/ml).Results. 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. Conclusion/interpretation. 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(+)- and Ca(2+)-ATPase are the major ATP-dependent membrane-bound enzymes that regulate the cation transmembrane gradient which is altered both in red blood cell (RBC) senescence and in RBCs of diabetic patients. In an attempt to clarify the possible connection between diabetes mellitus and ageing, we investigated the relationship between RBC ATP content, Na+/K(+)-ATPase, Ca(2+)-ATPase activities and ageing in healthy, insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) subjects. A significant correlation was found (r = -0.82; P < 0.001) between RBC ATP content and subject's age only in the control group. A significant reduction in Na+/K(+)-ATPase activity was observed in the older group (C2) of control subjects, in comparison with the younger (C1) one. In both IDDM and NIDDM subjects, the enzymatic activity was significantly decreased when compared with health subjects of similar age (P < 0.001). A significant negative correlation was found between age and enzymatic activity in healthy subjects (r = -0.60; P < 0.001). No difference was observed in the RBC membrane Ca(2+)-ATPase activity between younger (C1) and older (C2) healthy subjects. Ca(2+)-ATPase activity was significantly increased both in IDDM patients compared with C1 (P < 0.001) and in NIDDM patients compared with C2 (P < 0.001). The present data indicate that ageing causes a reduction in the erythrocyte ATP content in both healthy and diabetic subjects. In diabetic patients Na+/K(+)-ATPase activity decreases independently of age.