Fig. 33. EPR spectra of 2.03 complexes formed in vivo in mouse liver (curve a), in this tissue after homogena-tion (curve b), in supernatant fraction of liver homogenate (curve c) or in liver homogenate after treatment with CN- (curve d). Recordings at 77 K .
hexamethanol (Fig. 7) . This makes it plausible that matrices like phospholipids, fatty acids, etc. (compounds X) can also reduce the symmetry of protein-bound DNIC formed in tissues. DNIC with rhombic g-factors are observed in whole mouse liver tissue, liver homogenates and in supernatant fraction of the latter (Fig. 33) .
This points to a low-molecular nature of the compound's (compounds X) influence on the DNIC in the liver tissue. Interestingly, the dialysis of mouse blood plasma containing DNIC-BSA against mouse liver homogenates led to a sharp change in the shape of the EPR signal from plasma: it was similar to that from liver (Fig. 34) . Evidently, this transformation was due to the incorporation of low-molecular compounds X into DNIC-BSA. Consequent addition of DDS to the preparation increased the symmetry of the complex to axial one (Fig. 34) .
It cannot be excluded that species specificity of the EPR lineshape of the DNIC from liver of mouse, rat, guinea pig or cat mentioned before (Fig. 32) is due to the specificity of these intracellular low-molecular compounds affecting the DNIC structure. The transformation to axial symmetry of DNIC from mouse liver was also achieved by the addition of surface-active compound, Na-DDS to liver homogenates .
In the 1970s, it was discovered that nitrite anions induce the formation of DNIC in animal tissues and microorganisms [10-12,120-122]. The appearance of DNIC was attributed to acidification of nitrite with subsequent release of free NO molecules from nitrous acid [120-122]. However, recently it was found that various heme-iron proteins and xanthine oxidase have the capacity to reduce nitrite to NO (see Chapters 14-16 of this book).
Fig. 34. Shape of the EPR signals of DNICs formed in vivo in mouse liver (curve a) or mouse blood plasma (curve b) after adding DNIC with phosphate to mice or plasma, respectively. (curve c) EPR signals from blood plasma after dialysis against the suspension of mouse liver homogenate preparation. (curve d) The preparation (c) was treated with dodecyl sulfate (SDS). Recordings at 77 K (left side) or ambient temperature (right side) .
Although the mechanisms remain controversial, hypoxia or anoxia seems a necessary condition for the reduction of nitrite at physiological pH. The new data show that nitrite should also be considered as a source of NO in vivo.
It is important to distinguish between acute and long-term effects of nitrite. Large quantities of DNIC complexes appear in animal tissues only if the animals are maintained on nitrite diet for days. Bolus addition of nitrite to the animals leads mainly to the formation of NO-Hb complexes with the tissue yields of DNIC remaining far lower than in animals on a long-term nitrite-rich diet . The reason for this difference remains obscure. Without doubt, the elucidation of this problem will shed light on the mechanism of DNIC formation from nitrite.
The preceding discussion may have left the reader under the impression that nitrite be the dominant agent for the formation of DNIC complexes in tissues. However, it should not be forgotten that significant quantities of DNIC may also form from free NO radicals released by the enzymatic L-arginine dependent pathway. This pathway was confirmed by numerous investigators in a wide range of experiments on cultured animal and human cells [16-35]. However, the formation of the complexes in animal tissues was managed to be observed only several days after bacterial invasion artificially enhanced the NO synthesis by the inducible form of NO synthase . In normal conditions or in acute experiment with activation of existing constitutive NO synthases or enhanced synthesis by inducible NO synthase (iNOS), the levels of DNIC remained below the detection limit.
This raises the question why the endogenous DNIC remains so low in vivo. One reason may be the continuous destruction of the DNIC by endogenous peroxynitrite and superoxide
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