NITRIC-OXIDE SYNTHESIS IS IMPAIRED IN GLUTATHIONE-DEPLETED HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS

Human endothelial cells cultured from umbilical vein (HUVEC) were test ed for their ability to synthesize nitric oxide (NO), which has been i dentified as an endothelium-derived relaxing factor. The synthesis of this free radical (detected as citrulline, which is produced stoichiom etrically with NO from arginine) in HUVEC is Ca2+ dependent, is increa sed sevenfold by the calcium ionophore ionomycin, and accounts for mos t basal and ionomycin-induced guanosine 3',5'-cyclic monophosphate (cG MP) production. Loading of cells with reduced glutathione (GSH), but n ot with N-(2-mercaptopropionyl)-glycine (MPG), led to increased citrul line production, both basally and after ionomycin stimulation. When th e cells were depleted of GSH by incubation with 1-chloro-2,4-dinitrobe nzene (CDNB), citrulline synthesis and cGMP production were inhibited in a concentration-dependent way. CDNB was not cytotoxic and did not i nhibit cGMP increase elicited by sodium nitroprusside; cell loading wi th GSH (but not with MPG) relieved the block of citrulline synthesis. These results suggest that GSH is necessary in HUVEC for NO synthesis rather than for the NO effect on guanylate cyclase.