S-nitrosoglutathione efflux in the erythrocyte

Glutathione is an abundant molecule inside erythrocyte, originating S-nitrosoglutathione (GSNO) by reacting with nitric oxide (NO). GSNO has been regarded as a store and transporter of NO, with significant interest as a potential therapeutic agent, acting as an NO donor.NO metabolism inside the erythrocyte generates several derivatives, which can be altered by external and internal stimuli such as acetylcholine (ACh), a natural substrate of acetylcholinesterase (AChE). In spite of the knowledge gained in the last decades concerning NO efflux in erythrocytes little is known regarding erythrocyte GSNO efflux, which has also a significant role in microcirculation. Hence, the objective of this research was to evaluate the efflux of GSNO, concomitant with the efflux of NO, after stimulation with AChE effectors. To achieve these goals, the in vitro effect of AChE modulators - ACh and timolol - in erythrocyte NO and GSNO were studied. Timolol is an erythrocyte AChE inhibitor. Venous blood samples were collected from 18 healthy Caucasian men. For each blood sample, erythrocyte suspensions were obtained and incubated in the absence (controls) and presence of ACh and timolol maleate (10 μM final concentration of each modulator). Both timolol and ACh induced significant GSNO efflux in the erythrocyte when compared to the control; however the efflux was lower in the presence of timolol compared to ACh. Although erythrocyte NO efflux in presence of timolol is similar to the control, the efflux decreased when compared to the ACh treatment. The presence of timolol induces significant decrease of intra-erythrocyte GSNO levels, relative to control and ACh treatment. In conclusion, when erythrocytes were stimulated with ACh or timolol, GSNO efflux occurred associated with NO efflux. These new results bring new insight into the metabolism of erythrocyte NO and new possible therapeutic applications for GSNO.