Modulation of erythrocyte hemorheological properties by band 3 phosphorylation and dephosphorylation.

The objective of the present work was to study the effects of erythrocyte proteins phosphorylation in erythrocyte aggregation and deformability. Human whole blood samples were incubated in vitro in absent and in presence of the phosphorylation/dephosphorilation band 3 inhibitors and also with adenylyl cyclase, guanylate cyclase and PI3K inhibitors and the erythrocyte aggregation index (AIE) and deformability were assayed. The results show that when band 3 is phosphorylated in presence of a PTP inhibitor an increase in erythrocyte aggregation index is observed (p<0.0001). A partial dephosphorylation band 3 state, induced by PTK inhibitors, show a decrease in the erythrocyte aggregation index (p<0.002). However both manipulated states induced lower EAI values than blood samples aliquots controls. The guanylate cyclase and PI3-K inhibitors significantly decrease the erythrocyte aggregation index in relation with the control blood samples. Erythrocyte deformability in presence of all the inhibitors did not showed significant changes. PTP and PI3-K inhibitors showed a significantly increase in the plasma potassium concentrations not associated with EAI values. Methehemoglobin levels were increased significantly when guanylate cyclase inhibitor is present in the blood samples. In conclusion, the results suggest that erythrocyte aggregation index is dependent of the phosphorylated/dephosphorylated state of band 3.

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase.

Acetylcholinesterase is an enzyme whose best-known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X-100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X-100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C-terminal anti-acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N-terminal anti-acetylcholinesterase IgG antibody revealed a double band around 66-70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane-bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.

Acetylcholine and choline effects on erythrocyte nitrite and nitrate levels.

Acetylcholine has been detected in human blood. Acetylcholine receptors and acetylcholinesterase are present in erythrocyte membranes. We tested the acetylcholine and choline effects on nitric oxide metabolites (NOx), namely nitrites and nitrates, and observed if they are dependent on interactions with muscarinic receptors and acetylcholinesterase. Human erythrocyte suspensions were incubated with acetylcholine and choline in the absence or presence of 10 microM atropine or 10 microM velnacrine maleate. The nitrite and nitrate concentrations were determined by the Griess method. Acetylcholine or choline increased NOx control concentrations (P <0.001). The nitrite concentrations decreased in the presence of atropine or velnacrine maleate (P <0.03). The nitrate concentrations only decreased when velnacrine maleate was incubated with acetylcholine or choline (10 microM, P <0.03). These results demonstrated that acetylcholine and choline modulate nitric oxide metabolites on erythrocytes and this effect is mediated by interactions with erythrocyte membrane muscarinic receptors and membrane enzyme acetylcholinesterase. A hypothesis for the signal transduction mechanism has been discussed for acetylcholinesterase and muscarinic receptor (M1) participation.

Amperometric measurements of nitric oxide in erythrocytes.

In the recent years, there has been an increase in the development of new biosensors that could be helpful in the study of various physiological processes. In this study, we report the development of a new in vitro experimental design for real-time nitric oxide (NO) amperometric measurements in erythrocyte suspensions. To achieve this, we employed human erythrocyte suspensions in sodium chloride 0.9%, pH 7 (haematocrit 0.05%). The production of NO by erythrocytes was measured with a commercial NO sensor during stimulation by L-arginine, acetylcholine, choline, atropine and velnacrine maleate (10 microM of final concentrations). We also measured the nitrite and nitrate concentrations produced by erythrocyte suspensions stimulated with the above effectors by means of the Griess reaction method. We observed that there was a direct relation between the electric current produced by the NO sensor, and the NO standard concentrations, thereby leading to a good calibration curve. The in vitro erythrocytes produced significant amperometric NO values in response to a wide range of effectors and these results have the same variation profile of the nitrites and nitrates results achieved with the Griess method. In conclusion, the amperometric NO sensor constitutes a reliable method for direct, and real-time measurement in vitro of the NO production of erythrocyte suspensions, As such, it offers a potential diagnostic technique for the evaluation of diseases, and the therapeutic progression of diseases, related to intracellular NO metabolism.