Cellular antioxidant and pro-oxidant actions of nitric oxide.

We describe a biphasic action of nitric oxide (NO) in its effects on oxidative killing of isolated cells: low concentrations protect against oxidative killing, while higher doses enhance killing, and these two effects occur by distinct mechanisms. While low doses of NO (from (Z)-1-[N-(3-ammonio propyl)-N-(n-propyl)-amino]-diazen-1-ium-1,2(2) diolate [PAPA/NO] or S-nitroso-N-acetyl-L-penicillamine [SNAP] prevent killing of rat hepatocytes by t-butylhydroperoxide (tBH), further increasing doses result in increased killing. Similar effects occur with rat hepatoma cells treated with PAPA/NO and tBH or H2O2. Increased killing with higher concentrations of NO donor is due to both NO and tBH, because NO donor alone is without effect. Glutathione (GSH) is not involved in either of these actions. Based on measurements of thiobarbituric acid-reactive substances (TBARS) and effects of lipid radical scavenger (DPPD) and deferoxamine, the protective effect, but not the enhancing effect, involves peroxidative chemistry. Fructose has no effect on tBH killing alone but provides substantial protection against killing by higher concentrations of NO plus tBH, suggesting that the enhancing effect involves mitochondrial dysfunction. Hepatocytes, when stimulated to produce NO endogenously, become resistant to tBH killing, indicative of the presence of an NO-triggered antioxidant defensive mechanism. The finding that the protective effects of low concentrations of NO and the harmful effects of high concentrations of NO are fundamentally different in nature suggest that therapeutic interventions could be designed, which selectively prevent its pro-oxidant activity at high concentrations, thus converting NO from a "Janus-faced" modulator of oxidant injury into a "pure" protectant.

Seasonal changes in the production of two novel and abundant ovarian steroids in the channel catfish (Ictalurus punctatus).

Timely and appropriate changes in steroid plasma titers are necessary for successful reproduction in all vertebrates. Gonadal steroidogenesis of the most intensively cultured teleost species in North America, the channel catfish (Ictalurus punctatus), is poorly understood so a year-long study was conducted to investigate seasonal changes in ovarian steroidogenesis. Incubations of ovarian tissue were conducted monthly with [3H]pregnenolone and the medium was analyzed by high-performance liquid chromatography (HPLC) with radioactivity detection. The suite of steroids produced by the catfish ovary included the expected sex steroids (estradiol and testosterone) and 18 additional ovarian metabolites, including five steroids that have yet to be identified. Androstenedione, 20beta-dihydroprogesterone, 5|P-dihydrotestosterone, estriol, 11beta-hydroxyandrostenedione, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11beta-hydroxytestosterone, and progesterone were characterized by a combination of HPLC and thin-layer chromatography. Two of the most abundant steroids were isolated and analyzed by gas chromatography coupled with mass spectrometry (GC-MS). One of the steroids, 7alpha-hydroxypregnenolone (7P5), is a novel steroid in teleosts and was produced late in vitellogenic growth of the oocyte. Evidence suggests that the enzyme responsible for converting pregnenolone to 7P5, 7alpha-hydroxylase, is a cytochrome P450. The second abundant steroid metabolite was partially characterized by GC-MS as an hydroxylated form of 17-hydroxy-pregnenolone (chi,17P5). This steroid was most abundant when the ovary was regressed and during early vitellogenesis and rapidly decreased prior to spawning. In mammals, 7P5 has been identified as an important neurosteroid; however, the reproductive significance of 7P5 and chi,17P5 in catfish is unknown.

Activation of tumor necrosis factor-alpha-converting enzyme-mediated ectodomain shedding by nitric oxide.

Ectodomain shedding of cell surface proteins is an important process in a wide variety of physiological and developmental events. Recently, tumor necrosis factor-alpha-converting enzyme (TACE) has been found to play an essential role in the shedding of several critical surface proteins, which is evidenced by multiple developmental defects exhibited by TACE knockout mice. However, little is known about the physiological activation of TACE. Here, we show that nitric oxide (NO) activates TACE-mediated ectodomain shedding. Using an in vitro model of TACE activation, we show that NO activates TACE by nitrosation of the inhibitory motif of the TACE prodomain. Thus, NO production activates the release of cytokines, cytokine receptors, and adhesion molecules, and NO may be involved in other ectodomain shedding processes.