Velnacrine

Acetylcholinesterase (AChE) is found both on the membranes of neuronal and non-neuronal cells. In this study we performed intravenous administrations of velnacrine (VLN) and acetylcholine (ACh), respectively, AChE inhibitor and substrate, in an animal model of lipopolysaccharide (LPS)-induced inflammation in Wistar rats. Using intravital microscopy the number of rolling and adherent leukocytes in post-capillary venules was monitorized and blood samples were collected for TNF-α plasma concentrations determination. Our results showed that in presence of LPS, ACh has an anti-inflammatory effect, seen by a decrease in TNF-α plasma levels and maintains the number of rolling and adherent leukocytes. The presence of VLN before LPS almost blocked the LPS-induced rolling and TNF-α releasing. Thereby VLN seems to have, like ACh, an anti-inflammatory effect by diminishing TNF-α concentrations.

Circulating acetylcholine, substrate of membrane acetylcholinesterase (AChE), is known to enhance the band 3 protein degree of phosphorylation. The purpose of this study was to verify whether the band 3 phosphorylation status is associated with a G protein and whether it is an influent factor on AChE enzyme activity. From blood samples of healthy donors, erythrocyte suspensions were prepared and incubated with AChE substrate (acetylcholine) and inhibitor (velnacrine), along with protein tyrosine kinase (PTK) and tyrosine phosphatase (PTP) inhibitors. AChE activity was determined by spectrophotometry and extract samples were analyzed by western blotting using primary antibodies to different G protein subunits. Our results with phosphorylated band 3 (PTP inhibitor) show an increase in erythrocyte AChE (p < 0.0001). A dephosphorylated band 3 state (PTK inhibitor) shows a significant decrease. We identified a potential linkage of protein subunits Galpha(i1/2) and G(beta) with band 3 protein. Galpha(i1/2) and G(beta) may be linked to the band 3 C-terminal site. Galpha(i1/2) is associated with the band 3 N-terminal domain, except for the control and ACh aliquots. G(beta) is associated with both phosphorylated and dephosphorylated band 3 in the presence of velnacrine. We conclude that an erythrocyte G protein with subunits Galpha(i1/2) and G(beta) is associated with band 3. AChE depends on the degree of band 3 phosphorylation and its association with Galpha(i1/2) and G(beta).