Non-neuronal cholinergic system and signal transduction pathways mediated by band 3 in red blood cells

Abstract

Background: Non-neuronal acetylcholine (ACh) and acetylcholinesterase (AChE) have been recognized in the past. Vascular ACh has been associated by us with the regulation of microcirculatory flow by modulating nitric oxide (NO) intracellular mobilization, metabolism (NO_x) and release from erythrocytes, as well as the glycolytic flux. Velnacrine 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 translocation among nitrosylated molecules and phosphorylated/dephosphorylated states of band 3 protein, processed by major tyrosine-kinases (PTK: p72^syk, p53/56^lyn and p59/61^hck) and phosphotyrosine-phosphatases (PTP).

Methods: To assess this hypothesis under the influence of AChE effectors (acetylcholine/velnacrine), blood samples from healthy donors were harvested and Western blot analysis was subsequently used to determine the degree of band 3 phosphorylation, in the presence and absence of PTK/PTP inhibitors. NO and nitrites/nitrates were quantified using an amperometric method and the Griess Reaction, respectively, in erythrocyte suspensions. Measurements of erythrocyte metabolites (2,3-bisphosphoglycerate; glyceraldehyde 3-phosphate dehydrogenase; glucose-6-phosphodehydrogenase; lactate), hemoglobin and cyclic nucleotides were conducted afterwards.

Results: Increased levels of phosphorylated-band 3 obtained upon p72^syk inhibition suggest p59/61^hck and p53/56^lyn as secondary involved kinases. As to NO/NO_x quantification, in the presence of PTK_i we reported higher levels with 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, glyceraldehyde 3-phosphate dehydrogenase and glucose-6-phosphodehydrogenase were found to decrease with ACh, whereas P50, lactate and both cGMP/cAMP happened to increase.

Conclusion: Changes on human erythrocyte NO(x) 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 events may potentially change under pathological conditions, coadjuvant drugs could become accessible in the setting of microcirculation disease.