Modulation of Erythrocyte Acetylcholinesterase Activity and Its Association with G Protein-Band 3 Interactions

Abstract

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).

Physiological properties of human erythrocytes in inflammation

“…It was verified that the AChE-ACh enzyme active complex activate the protein kinase C (PKC) which in turn phosphorylate the protein tyrosine kinase (PTK) turning it from inactive to active state. PTK phosphorylate band 3 protein became able to receive NO in its thiol group from S-nitrosohemoglobin for delivery to extracellular medium [9][10][11][12]. PKC inhibited by phosphorylation protein tyrosine phosphatase (PTP) and also the phosphodiesterase 3 (PDE3) which consequently do not hydrolyzes the cyclic adenosine triphosphate (cAMP) [9,10].…”

“…PKC inhibited by phosphorylation protein tyrosine phosphatase (PTP) and also the phosphodiesterase 3 (PDE3) which consequently do not hydrolyzes the cyclic adenosine triphosphate (cAMP) [9,10]. The AChE-ACh enzyme active complex joined to G␣i protein which inhibit adenylyl cyclase (AC) became it unable to cAMP formation form ATP [11]. Active AChE enzyme conformations are associate with the increase of NO efflux from erythrocyte, which is further potentiate by ACh [12].…”

“…The cAMP levels did not interfere. This is the scientific rational base of the efficient methodology for estimate erythrocyte bioavailability in NO [7][8][9][10][11][12].…”

Fibrinogen Effects of Erythrocyte Nitric Oxide Metabolism in Presence of Timolol

“…Maintaining high Fib concentrations, the levels of NO efflux from RBCs normalize by the presence of acetylcholine (ACh) binding to RBCs membrane acetylcholinesterase (AChE) [8]. The explanation comes from the signal transduction pathway initiate by the AChE-ACh active complex forms [9,10]. So, activation of protein kinase C (PKC) inhibits by phosphorylation the phosphodiesterase (PDE3) resulting increased amount of cAMP concentration that abolish the higher NO efflux induced by hyperfibrinogenemia observed in absence of ACh [8][9][10].…”

“…The explanation comes from the signal transduction pathway initiate by the AChE-ACh active complex forms [9,10]. So, activation of protein kinase C (PKC) inhibits by phosphorylation the phosphodiesterase (PDE3) resulting increased amount of cAMP concentration that abolish the higher NO efflux induced by hyperfibrinogenemia observed in absence of ACh [8][9][10]. While ACh increase erythrocyte deformability (ED) at high shear stress, high Fib concentrations increase it at low shear stress under band 3 dephosphorylated by protein tyrosine kinase (PTK syk) inhibition [11,12].…”