Patrícia Napoleão

Acetylcholinesterase Conformational States Influence Nitric Oxide Mobilization in the Erythrocyte

In the human erythrocyte, band 3 protein mediates nitric oxide (NO) translocation and its effects are strongly related to phosphorylated/dephosphorylated intracellular states. The metabolism of NO could change in the presence of acetylcholinesterase (AChE). Therefore, the present study was designed to assess the effect of conformational changes in AChE (via N-19 and C-16 antibodies) and enzymatic inhibition/activation of protein kinase C (PKC) in erythrocyte NO mobilization in vitro. Our results show that by inhibiting PKC with cheletrine, impaired erythrocyte NO efflux and s-nitrosoglutathione (GSNO) levels were verified, while PKC’s activation by Phorbol 12-myristate 13-acetate had the opposite effect. Those results demonstrate the influence of 4.1R complex and band 3 protein level of phosphorylation on NO efflux and GSNO concentration mediated by PKC inhibition/activation. In addition, the present study shows evidence that conformational changes in AChE promoted by incubation with N-19 and C-16 antibodies alter the enzymes functional connection to acetylcholine (ACh) (AChE-ACh complex) in an irreversible manner, resulting in impaired GSNO concentration and NO efflux from the erythrocyte. Novel insight into NO metabolism in the erythrocyte is brought with the presented findings allowing new possibilities of modulating NO delivery, possibly involving PKC and AChE conformational alterations in combination.

S-nitrosoglutathione efflux in the erythrocyte.

Glutathione is an abundant molecule inside erythrocyte, originating S-nitrosoglutathione (GSNO) by reacting with nitric oxide (NO). GSNO has been regarded as a store and transporter of NO, with significant interest as a potential therapeutic agent, acting as an NO donor.NO metabolism inside the erythrocyte generates several derivatives, which can be altered by external and internal stimuli such as acetylcholine (ACh), a natural substrate of acetylcholinesterase (AChE). In spite of the knowledge gained in the last decades concerning NO efflux in erythrocytes little is known regarding erythrocyte GSNO efflux, which has also a significant role in microcirculation. Hence, the objective of this research was to evaluate the efflux of GSNO, concomitant with the efflux of NO, after stimulation with AChE effectors. To achieve these goals, the in vitro effect of AChE modulators - ACh and timolol - in erythrocyte NO and GSNO were studied. Timolol is an erythrocyte AChE inhibitor. Venous blood samples were collected from 18 healthy Caucasian men. For each blood sample, erythrocyte suspensions were obtained and incubated in the absence (controls) and presence of ACh and timolol maleate (10 μM final concentration of each modulator). Both timolol and ACh induced significant GSNO efflux in the erythrocyte when compared to the control; however the efflux was lower in the presence of timolol compared to ACh. Although erythrocyte NO efflux in presence of timolol is similar to the control, the efflux decreased when compared to the ACh treatment. The presence of timolol induces significant decrease of intra-erythrocyte GSNO levels, relative to control and ACh treatment. In conclusion, when erythrocytes were stimulated with ACh or timolol, GSNO efflux occurred associated with NO efflux. These new results bring new insight into the metabolism of erythrocyte NO and new possible therapeutic applications for GSNO.

Timolol Modulates Erythrocyte Nitric Oxide Bioavailability

Changes of oxygen partial pressure in tissues are sensed by the erythrocytes that with the efflux or with the maintenance of nitric oxide that contains promote vasodilation or vasoconstriction. Binding of acetylcholine with the acetylcholinesterase of the erythrocyte membrane originates a signal transduction mechanism that involves both the Gi protein as well as band 3 protein what stimulates nitric oxide efflux. The bioavailability of nitric oxide in presence of velnacrine maleate an inhibitor of acetylcholinesterase is preserved what means that there are no changes in the nitric oxide efflux. Timolol maleate is an inhibitor of acetylcholinesterase.The aim of this study was to assess the role of the timolol maleate in the erythrocyte in respect to bioavailability on nitric oxide and compare it with effect resulting of the presence of acetylcholine. Venous blood samples were collected from the forearm vein of fifteen healthy Caucasian men after informed consent. Each blood sample was divided in three 1 mL samples, centrifuged, and suspensions of erythrocyte were performed in order to achieve 10 μM final concentration either of acetylcholine or of the timolol. Levels of nitric oxide efflux were evaluated by amperometric method. S-nitrosoglutathione, nitrites and nitrates were assessed with the Griess reaction using the spectrophotometric method.The nitric oxide efflux by the erythrocyte in presence of timolol is like to the control sample but significantly decreased it when compared to the sample with acetylcholine. The presence of timolol induces decrease in the erythrocyte levels of S-nitrosoglutathione significantly in relation with the control and with acetylcholine samples.In conclusion, in vitro, in erythrocyte the nitric oxide content is maintained by timolol maleate. It may be expected the same role for timolol in the ocular microcirculation when being applied as a therapeutic compound.

Changes of soluble CD40 ligand in the progression of acute myocardial infarction associate to endothelial nitric oxide synthase polymorphisms and vascular endothelial growth factor but not to platelet CD62P expression

Reported in vitro data implicated soluble CD40 ligand (sCD40L) in endothelial dysfunction and angiogenesis. However, whether sCD40L could exert that influence in endothelial dysfunction and angiogenesis after injury in acute myocardial infarction (AMI) patients remains unclear. In the present study, we evaluated the association of sCD40L with markers of platelet activation, endothelial, and vascular function during a recovery period early after AMI. To achieve this goal, the time changes of soluble, platelet-bound, and microparticle-bound CD40L levels over 1 month were assessed in AMI patients and correlated with endothelial nitric oxide synthase (eNOS) polymorphisms, vascular endothelial growth factor (VEGF) concentrations, and platelet expression of P-selectin (CD62P). The association of soluble form, platelet-bound, and microparticle-bound CD40L with CD62P expression on platelets, a marker of platelet activation, was also assessed to evaluate the role of CD40L in the thrombosis, whereas the association with eNOS and VEGF was to evaluate the role of CD40L in vascular dysfunction. This work shows for the first time that time changes of sCD40L over 1 month after myocardial infarct onset were associated with G894T eNOS polymorphism and with the VEGF concentrations, but not to the platelet CD62P expression. These results indicate that, in terms of AMI pathophysiology, the sCD40L cannot be consider just as being involved in thrombosis and inflammation but also as having a relevant role in vascular and endothelial dysfunction.

Crosstalk between inflammation, iron metabolism and endothelial function in Behçet’s disease

Behçets disease (BD) is a rare chronic vasculitis of unclear etiology. It has been suggested that inflammatory response has an important role in BD pathophysiology. Herein, we aimed to study the interplay between inflammation, iron metabolism and endothelial function in BD and search for its putative association with disease activity. Twenty five patients clinically diagnosed with BD were selected and twenty four healthy age-sex matched individuals participated as controls. Results showed an increase of total number of circulating white blood cells and neutrophils, serum transferrin, total iron binding capacity, mieloperoxidase (MPO), ceruloplasmin (Cp), C reactive protein, β2 microglobulin and Cp surface expression in peripheral blood monocytes in BD patients comparatively to healthy individuals (p < 0,05). Of notice, the alterations observed were associated to disease activity status. No significant differences between the two groups were found in serum nitric oxide concentration. The results obtained suggest an important contribution from innate immunity in the pathogenesis of this disease. In particular, surface expression of leukocyte-derived Cp may constitute a new and relevant biomarker to understand BD etiology.

Systemic markers of the redox balance and apolipoprotein E polymorphism in atherosclerosis: the relevance for an integrated study.

Prospective studies have demonstrated that an imbalance between oxidative damage and antioxidative protection can play a role in the development and progression of atherosclerosis. Also, genotypes with the apolipoprotein E ζ4 allele have been associated with an increase risk for this pathology. Based on this knowledge, the aim of this study was to evaluate indicators of the redox balance, trace elements, and apolipoprotein E allelic profile in subjects from the Lisbon population with clinically stable atherosclerosis, at risk for atherosclerotic events, and in healthy subjects for comparison. The activities of superoxide dismutase in erythrocytes and glutathione peroxidase in whole blood, plasma total thiols, and serum ceruloplasmin were kept unchanged among the three groups. Serum α-tocopherol was increased in atherosclerotic patients. Total malondialdehyde in serum and protein carbonyls in plasma, which are indicators of lipid and protein oxidative damage, respectively, reached their highest values in risk subjects. The concentrations of potassium and calcium, in plasma and in blood cells, were slightly elevated in patients and might reflect an electrolytic imbalance. Regarding the apolipoprotein E polymorphism, atherosclerotic patients had an increased incidence of the high-risk genotypes for atherogenesis (ζ3/ζ4 and ζ4/ζ4). A multivariate model applied to the general population using most of the parameters clearly separated the three groups at study (i.e., the healthy group from the steady-state group of risk disease and from the atherosclerotic one). As shown by us, the usefulness of biochemical and complementary genetic markers is warranted for a better knowledge on atherosclerosis molecular basis.

Erythrocyte nitric oxide in glaucoma patients : ex vivo study

BACKGROUND Glaucoma is an optic neuropathy associated with vascular dysregulation and increased intra-ocular pressure (IOP). Timolol is used as treatment for reducing IOP, by limiting aqueous humour production. Increased NOS expression as well as decreased levels of nitric oxide (NO) metabolites, and high activity of erythrocyte acetylcholinesterase (AChE) were observed in primary open angle glaucoma patients. OBJECTIVE This ex vivo study aims to evaluate timolol effect in NO efflux and its derivatives in glaucoma patients erythrocytes. METHODS Venous blood from 15 glaucoma patients was collected. Erythrocyte suspensions were incubated with the AChE modulators acetylcholine (ACh) and timolol at 10 μM. Erythrocyte NO efflux and S-nitrosoglutathione (GSNO) concentration were measured. RESULTS No significant differences were obtained in erythrocyte NO efflux and GSNO concentration in response to ACh or timolol when compared with the untreated erythrocytes of glaucoma patients. When comparing the same incubation conditions for erythrocyte suspensions between glaucoma patients and healthy subjects, those from glaucoma patients showed higher NO efflux in presence and absence of timolol, and higher values of GSNO in the presence of timolol. CONCLUSIONS We demonstrated that erythrocytes from glaucoma patients have similar availability to release NO both in absence and presence of timolol, and have higher GSNO values in presence of timolol.

Identification of erythrocyte biomarkers in amyotrophic lateral sclerosis.

INTRODUCTION Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. It has been hypothesised that red blood cells (RBCs) may be involved in the disease process by the release of damaging molecules. OBJECTIVE The aim of this ex vivo study is to compare RBCs biochemical and hemorheological parameters between ALS patients and healthy donors to identify novel biomarkers of the ALS disease. METHODS We included 82 ALS patients and 40 gender age-matched healthy donors. We performed quantification of erythrocyte aggregation and deformability, nitric oxide (NO) efflux from RBCs, acetylcholinesterase (AChE) enzyme activity and intraerythrocytic concentration of nitrite, nitrate and S-nitrosogluthatione (GSNO). RESULTS Erythrocyte deformability and AChE activity were increased in patients with ALS in comparison to healthy donors. NO efflux from RBCs and concentration of intraerythrocytic nitrite were lower in ALS patients. In patients, we found that for higher NO range of values the respiratory function is worse and that for higher AChE range of values the RBCs nitrite content increase. CONCLUSION The results of the present study indicate that NO efflux from RBCs and RBCs AChE should be further explored as potential biomarkers for ALS.

An ex vivo study of nitric oxide efflux from human erythrocytes in both genders.

INTRODUCTION Acetylcholinesterase (AChE) is located on outer surface of erythrocyte membrane. Gender-related differences in erythrocyte AChE enzyme activity had been verified in young adults. It is also known that binding of acetylcholine (ACh) with AChE on erythrocyte membrane initiates a signal transduction mechanism that stimulates nitric oxide (NO) efflux. AIMS This ex vivo study was done to compare the amount of NO efflux obtained from erythrocytes of healthy donors in males and females. METHODS We included 66 gender age-matched healthy donors (40-60 years old). We performed quantification of erythrocyte NO efflux from erythrocytes and of the membrane AChE enzyme activity. RESULTS There are no significant differences in NO efflux from erythrocytes between men and women. Regarding AChE enzyme activity values, in this range of age, no differences between genders were obtained. However, the values of AChE enzyme activity in the third quartile of NO efflux values were significantly higher (p < 0.05) in women than in men. CONCLUSIONS The efflux of NO from erythrocyte of healthy humans did not change with gender. For the same range of values of NO efflux from erythrocytes, in both gender, it was verified higher values of AChE enzyme activity in women.

T lymphocytes alterations are associated with oxidized LDL, troponin T, white blood cells and C-reactive protein during acute myocardial infarction.

BACKGROUND Auto-immune responses are associated with oxidized LDL (ox-LDL) release, a key factor in plaque destabilization. Data on the relationship between ox-LDL and T lymphocytes in human populations remains scarce. T cells also react with other molecules from the lesion and/or damage the myocardium. OBJECTIVE The objective of the present study was to examine the relationship between circulating T lymphocytes, ox-LDL, markers of myocardial necrosis (cTnT), myocardial dysfunction (N-terminal pro-brain natriuretic peptide - NT-proBNP) and inflammation (C-reactive protein - CRP) in the setting of acute myocardial infarction. METHODS A longitudinal study of 55 patients with ST-elevation myocardial infarction (STEMI) were evaluated at three time points: admission, 2 and 40 days following admission, together with 30 patients with stable angina (SA) and 56 subjects without coronary artery disease serving as controls (CTR). RESULTS STEMI patients had maximal ox-LDL values and minimal levels of CD3+ T lymphocytes at admission, which was normalized during the recovery period. The increasing trend of CD3+ T cells was positively associated with an ox-LDL decline over time. CRP and cTnT longitudinal variations were negatively associated with the CD3+ T-cell increasing trend. These associations were not found in SA patients or controls. CONCLUSIONS The associations found between CD3+ T lymphocytes, ox-LDL and cTnT suggest a specificity of the immune response in AMI towards arterial and myocardial inflammation and remodelling.