Fig. 8. Dose response curves for relaxation of precontracted rat aortic rings by acetylcholine (left panel) and GSNO (right panel). The presence of 0.3 mM pyrogallol (open triangles) cancels the stimulus from Ach, and reduces the sensitivity to GSNO by an order of magnitude. Experiments in Krebs buffer at 37°C at high oxygen tension (95%O2 + 5%CO2). (From Ref. [89].)

RAW264.7 macrophages contain a comparatively low amount of non-heme iron. These macrophages proved quite susceptible to the pro-apoptotic action of RSNOs. Preincubation with exogenous iron reinforced their resistance to apoptosis and EPR spectroscopy revealed the formation of intracellular DNIC with thiol-containing ligands [90]. These DNIC were evidently assembled from RSNO and non-heme iron. In contrast, hepatocytes contain fairly large amounts of endogenous iron and show much higher resistance to the pro-apoptotic effect of NO [90]. The cellular reaction pathways for DNIC are considered in detail in Chapter 11. Intracellular formation of DNIC was also observed after addition of ferrous iron to cultured leukemic cells incubated with S-nitrosocysteine (Vanin, A.F., non-published data). The incubation of cells with Cys-DNIC or GS-DNIC did not affect the cell viability.

The mechanism of apoptotic protection was investigated in cellular assays [90]. It was shown that apoptosis was suppressed by inhibition of caspase 3 by S-nitrosation of this protein. The S-nitrosation could be reversed by the addition of dithiothreitol. It demonstrated that extracellular DNIC enhanced S-nitrosation of the intracellular proteins. Details of the mechanism remain unclear since transport of intact DNIC across cellular membranes remains unproven at this moment.

We just noted that both RSNO and DNIC can act as NO donors in their own respect. Usually, activation of a physiological response requires higher doses of GSNO than DNIC [90,95]. As donors, they are clearly distinguished by their response to alterations in the pool of free iron and copper in the solutions. Addition of a metal chelator like bathophenanthroline disul-fonate (BPDS) will remove the iron from DNIC and induce instantaneous release of the nitrosyl ligands into the solution. The effect on GSNO is exactly opposite in that its lifetime is greatly extended by chelation of the free metal ions. But the pools of RSNO and

DNIC are always coupled in a dynamic equilibrium by the presence of free iron ([96] and Chapter 11). It should be noted that tissues always contain a certain quantity of loosely bound iron (experimental evidence for this iron pool in tissues is reviewed in Chapter 2).

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