It is well established that nitric oxide (•NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all •NO-derived adducts (900 pmol/mg protein (45–90 μM)). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics of, DNIC assembly and disappearance from endogenous and exogenous sources of •NO in relation to iron and O2 concentrations. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of •NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic •NO concentrations (50–500 nM). Decreasing the O2 concentration reduced the rate of enzymatic •NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t½ = 80 min) and remained detectable long after the •NO source was removed (>24 h). These results demonstrate that DNIC will be formed under all cellular settings of •NO production and that the contribution of DNIC to the multitude of observed effects of •NO must always be considered.

Three functional hERG channel assay methods have been developed and evaluated. The methods were tested against five known hERG channel inhibitors: dofetilide, terfenadine (Seldane), sertindole (Serdolect), astemizole (Hismanal), and cisapride (Propulsid). The DiBAC4(3)-based assays were found to be the most economical but had high false-hit rates as a result of the interaction of dye with the test compounds. The membrane potential dye assay had fewer color-quenching problems but was expensive and still gave false hits. The nonradioactive Rb+ efflux assay was the most sensitive of all the assays evaluated and had the lowest false-hit rate.

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