BACKGROUND: Non-neuronal acetylcholine (ACh) and acetylcholinesterase (AChE) have been recognized in t Vascular ACh has been associated by us with the regulation of microcirculatory flow by modulating nitric oxide ( intracellular mobilization, metabolism (NOx) and release from erythrocytes, as well as the glycolytic flux. Velnacri maleate is a well-known AChE inhibitor which plays a competitive role by decreasing NO-mediated erythrocyte responses. A plausible hypothesis to explain the mechanisms underlying those events hinges on the NO translo among nitrosylated molecules and phosphorylated/dephosphorylated states of band 3 protein, processed by maj tyrosine-kinases (PTK: p72syk, p53/56lyn and p59/61hck) and phosphotyrosine-phosphatases (PTP).
METHODS: To assess this hypothesis under the influence of AChE effectors (acetylcholine/velnacrine), blood sa from healthy donors were harvested and Western blot analysis was subsequently used to determine the degree phosphorylation, in the presence and absence of PTK/PTP inhibitors. NO and nitrites/nitrates were quantified usi amperometric method and the Griess Reaction, respectively, in erythrocyte suspensions. Measurements of eryth metabolites (2,3-bisphosphoglycerate; glyceraldehyde 3-phosphate dehydrogenase; glucose-6-phosphodehydro lactate), hemoglobin and cyclic nucleotides were conducted afterwards.
RESULTS: Increased levels of phosphorylated-band 3 obtained upon p72syk inhibition suggest p59/61hck and as secondary involved kinases. As to NO/NOx quantification, in the presence of PTKi we reported higher levels velnacrine-AChE, as opposed to acetylcholine-AChE. Calpeptin, a PTP inhibitor which triggers full band 3-phosphorylation, led to the opposite NO mobilization, being reinforced by ACh. Oxy-hemoglobin, glyceraldehy 3-phosphate dehydrogenase and glucose-6-phosphodehydrogenase were found to decrease with ACh, whereas lactate and both cGMP/cAMP happened to increase.
CONCLUSION: Changes on human erythrocyte NOx mobilization and metabolic fluxes occur under influence of non-neuronal ACh/AChE, in turn dependent on the degree of band 3-phosphorylation. Since these vascular eve potentially change under pathological conditions, coadjuvant drugs could become accessible in the setting of microcirculation disease.
Xanya Sofra Weiss
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