Acetylcholinesterase Conformational States Influence Nitric Oxide Mobilization in the Erythrocyte

Abstract

In the human erythrocyte, band 3 protein mediates nitric oxide (NO) translocation and its effects are strongly related to phosphorylated/dephosphorylated intracellular states. The metabolism of NO could change in the presence of acetylcholinesterase (AChE). Therefore, the present study was designed to assess the effect of conformational changes in AChE (via N-19 and C-16 antibodies) and enzymatic inhibition/activation of protein kinase C (PKC) in erythrocyte NO mobilization in vitro. Our results show that by inhibiting PKC with cheletrine, impaired erythrocyte NO efflux and s-nitrosoglutathione (GSNO) levels were verified, while PKC's activation by Phorbol 12-myristate 13-acetate had the opposite effect. Those results demonstrate the influence of 4.1R complex and band 3 protein level of phosphorylation on NO efflux and GSNO concentration mediated by PKC inhibition/activation. In addition, the present study shows evidence that conformational changes in AChE promoted by incubation with N-19 and C-16 antibodies alter the enzyme's functional connection to acetylcholine (ACh) (AChE-ACh complex) in an irreversible manner, resulting in impaired GSNO concentration and NO efflux from the erythrocyte. Novel insight into NO metabolism in the erythrocyte is brought with the presented findings allowing new possibilities of modulating NO delivery, possibly involving PKC and AChE conformational alterations in combination.

Physiological properties of human erythrocytes in inflammation

“…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].…”

“…CD47 which is a component of Rh group associate with G␣i protein that inhibiting AC decrease the level of cAMP [17]. When hyperfibrinogenemia is mimicked in vitro erythrocyte NO efflux increased in dependence of band3 protein phosphorylation returning to normal levels when in presence of either ACh or timolol (AChE inhibitor) showing dependency of the AChE enzyme conformational states [12,[18][19][20]. However if the AC completely inhibited by the specific inhibitor MDL, in presence of high fibrinogen levels and either timolol or 4N1K (a binding peptide of CD47) an increase of erythrocyte NO efflux is verified [21].…”

“…The levels of NO efflux return to normal with increased cAMP concentration achieved either by PDE3 enzyme dephosphorylated in inactive state (resulting from inhibition of PI3-K by worthamin) or by the AC activation by forskolin [21,22]. Those results allows us to conclude that in acute or chronic inflammation where soluble fibrinogen concentration are above the normal range, the erythrocyte ability to scavenger maintaining NO or deliver it is dependent of band 3 protein phosphorylation degree, low cAMP levels and AChE enzyme molecule conformations [12].…”

“…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].…”