Biomedical Engineering Reference
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Figure 3. Change of nitric oxide production (a) and virus reproduction (b) in vascular endothelial cells
after treatment with human interferon-α for 24 hours and subsequently infected with HSV-1 (MI, 0,1);
Reduction of NO production of IFN-treated and infected cultures can not be explained by
the action of IFN, since in the absence of infection IFN at concentrations 1x10
5
IU / ml and
1x10
6
IU / ml caused an increase of NO production in ECs (Figure 1). Effect of HSV-1 at low
MOI consisted in reducing nitric oxide synthesis by ECs (Figure 2). The connection between
low levels of virus reproduction by endothelial cells under the influence of IFN to 24 hours
(Figure 3a) and, consequently, low virus content, and decreased nitric oxide synthesis by
these cells to 24 hours was observed (3a). The increase in virus reproduction in cells treated
with IFN for 48 hours, and, consequently, a higher concentration of the virus, accompanied
by increased synthesis of nitric oxide in ECs (Figure 3b). The proposed mechanism of this
process indicates a complex relationship between the action of IFN-α, virus infection and NO
production by endothelial culture. Probably it is due to the overlapping intracellular signaling
mechanisms responsible for the effect of IFN-α and HSV on the expression of iNOS in
endothelial cells. Nitric oxide synthesized by endothelial cells in the incubation medium also
was detected by ESR. As the trap NO in this case, we used Hb, which has a high affinity for
NO. Figure 4 shows EPR spectra recorded in samples of cell medium in which untreated EC
were incubated for 24 h. Before preparing samples for EPR investigations into the incubation
medium were added Hb (5x10
-6
M) as a trap NO and AK (10
-5
M) as a reducing nitrite ions.
In the observed EPR spectrum a well-known signal of nitrosyl complexes of hemoglobin
with nitric oxide (NC Hb-NO) - a broad ESR signal with a characteristic triplet splitting at g-
factor of 2.01 was recorded. These data indicate that NO secreted by endothelial cells, getting
into the bloodstream, interacts with the molecules of Hb, displacing oxygen, which leads to
the formation of nitrosyl complexes Hb-NO in blood. This method can be used to test the NO
in the medium of cultured EC.
Thus, this study demonstrated that IFN-α activates nitric oxide production by cells of
primary culture of human ECs of blood vessels and the value of the effect was depended on
the concentration of the drug. Earlier, a similar concentration effect was observed in studying
of the action of IFN-γ on ECs. It was shown that IFN-γ at concentrations up 1 mg / ml led to a
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