Doubts concerning functional endothelial nitric oxide synthase in human erythrocytes

摘要

Nitric oxide (NO) is involved in the modulation of multiple physiologic functions. NO is produced from L-Arg by the catalytic action of NO synthase (NOS; EC 1.14.13.39). Erythrocytes have been reported to express NOS, an eNOS isoform. However, findings on a functional erythrocytic eNOS (eeNOS) in humans are contradictory.2"8 Discrepancies may be because of different experimental conditions and methodological shortcomings. In consideration of the paradoxical occurrence of eeNOS in red blood cells (RBCs),6 which are mainly responsible for NO inactivation, we attempted to measure NOS activity in RBCs freshly collected from normal healthy humans by a fully validated, highly sensitive and specific gas chromatography-mass spectrometry (GC-MS) assay. Approval from the local Ethics Committee of the Hannover Medical School was obtained. Blood was drawn from the antecubi-tal vein of healthy male and female volunteers using EDTA monovettes and processed immediately. RBCs were separated by centrifugation (800g, 4 deg C, 5 minutes) and used either unwashed or after repeated wash with physiologic saline. Unwashed RBCs were lyzed by freezing (30 minutes, -80 deg C) followed by slow defrosting in an ice bath and by rapid vortex-mixing (1 minute) with ice-cold distilled water (1:1, vol/vol).