Abstract
The requirements and influence of thiols on the production of nitric oxide (NO) were examined in cultured porcine aortic endothelial cells. NO production was diminished when cells were pretreated with thiol-depleting agents (IC50: N-ethylmaleimide, 30 microM; 1-chloro-2,4-dinitrobenzene, 200 microM; diamide, 1.5 mM; diethyl maleate, 20 mM). The depletion of glutathione (45-99% loss at the various IC50 values) and protein thiols (3-25% loss at IC50) showed no consistent relationship to decreased NO production. The effects of the agents on NO production were not linked to altered sensitivity to the stimulant (calcium ionophore A23187; maximal effect at 10 microM), but roughly paralleled the appearance of cell damage (17-44% lactate dehydrogenase release at IC50). The decrease in NO production due to 1-chloro-2,4-dinitrobenzene was partially reversed by cysteine, dithioerythritol, and dihydrolipoate, whereas cystine partially reversed the decrease due to diamide or diethyl maleate. On the other hand, several thiols diminished NO production in control cells. Overall, alterations of NO production did not parallel the depletion or replenishment of either glutathione, protein thiol, or soluble thiol pools, and so the results argue against hypotheses that cellular thiols are either substrates or necessary cofactors in the pathway of NO synthesis in endothelial cells.
Highlights
The requirements and influence of thiols on the production of nitric oxide (NO) were examined in cultured porcine aortic endothelial cells
Several arginine analogues, such aNs “-nitro-L-arginine (NNA) and Nu-monomethyl-L-arginine (LMMA), reversibly inhibit NO synthesis [4, 6], but little else is known about the chemistry of the enzyme
Since cellular thiol pools are tied to each other and to other metabolic pathways, the use of multiple types of thiol-altering agents are essential to distinguish a true involvement of a thiol pool inthe NO synthesis pathway from a coincidental relationship [13, 14]
Summary
+ Procedures” at theend of a subsequent 40-min incubation with either no supplementation, 1mM cysteine, or 1 mM cysteine 1mM BSO as indicated. The regardless of pretreatment (Fig. 5C), butit appeared that BSO addition of extracellular cysteine, which elevated the soluble lessened cysteine entry into cells. This confounding effect of thiol content of all cells (Fig. 5C),did not raise NO production, BSO has been attributed to its competitive inhibition of a but rather tended tlower it (Fig. 5A).The relatively efficient cysteine transport system [18]. There was usually an additional inhibition, rather than a agrees well with evidence that theglutathione S-conjugate of recovery, of NO production following addition of other types CDNB releases y-glutamylcysteine synthetase from feedback of thiols to thiol-depleted cells (Fig. 6A).
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