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C.N. Novikov, N.D. Vilenskaya, R.R. Asfaramov, O.A.Leontieva, and V.L. Voeikov
Faculty of Biology, Lomonosov Moscow State University, Moscow, Russia
Evidence is accumulating that generation of electron excited states (EES) as products of reaction with reactive oxygen species (ROS) permanently takes place in living organisms. Energy released due to EES relaxation may be transmitted from the sites of its generation (donors) to the sites of its utilization (acceptors) both by radiation-less and radiative mechanisms and play physiological role. It may be used for high quantum energy demanding processes, for energy pumping of macromolecular complexes. Electromagnetic fields created with these complexes and ensembles and their oscillatons may play informative role providing living system integrity.
The purpose of this work was the investigation of EES generation and their role in non-diluted human blood in comparison with the suspension of isolated neutrophils using the method of lucigenin- or luminol-dependent chemiluminescence (LG-CL and LM-CL, respectively). It is known, that LG-CL indicates of O2 generation in a system, while LM-CL is a probe for the generation of predominantly other reactive oxygen species (H2O2, OH , OCl , NO).
In freshly obtained healthy donor blood in the absence of neutrophil activators intensity of LG-CL was always much higher than that of LM-CL. After oxidative burst (OB) initiation in blood with zymosan both LG-CL and LM-CL were increasing. Blood dilution with physiological saline was followed with severe drop in LG-CL, while LM-CL continued to grow. On the contrary, in neutrophil suspensions LG-CL was always much lower than in non-diluted blood both in the presence and absence of OB initiator.
High intensity of LG-CL in fresh non-diluted healthy donors' blood indicates that leukocytes, and in particular neutrophils continuously generate O2. As practically all molecular oxygen in blood is bound to hemoglobin, leukocytes can get it only from erythrocytes. In fact, blood isolation from the ambient air did not diminish LG-CL levels. On the contrary, blood isolation from the air at the early stages of OB initiation blocked LM-CL intensity elevation, and it dropped down to negligible values. However, when LM-CL intensity reaches some particular level, cessation of blood contact with the air does not significantly change the time course of LM-CL development.
LG-CL dependence upon O2 supply from erythrocytes in non-diluted blood allowed to observe a new phenomenon related to the physiological function of CO (carbon monooxide, coal gas). CO is produced by heme-oxygenase in vessel endothelial cells and it is generally considered to regulate the activity of guanylate cyclase. However, this hypothesis overlooks that hemoglobin heme concentration is much higher than guanylate cyclase hemes in the vicinity of CO production and that CO affinity to hemoglobin 220-fold exceeds that of O2. Therefore CO should in the first place react with hemoglobin of erythrocytes. We found that introduction of several microliters of CO into native blood is followed with sharp and prolonged increase of LG-CL that immediately dropped down after 8-fold blood dilution with saline. On the contrary, when CO is passed through the neutrophil suspension both LG-CL and LM-CL intensity declines presumably due to the blockade by CO of heme-containing enzymes responsible for ROS production. Induction by CO of a sharp increase of LG-CL in non-diluted in contrast to diluted blood or isolated neutrophil suspensions may be explained by previously unknown ability of CO to induce massive O2 release from hemoglobin in erythrocytes possibly because each binding event of this gas molecule to a free hemoglobin molecule triggers O2-heme dissociation in many other hemoglobin molecules. These process allows to facilitate O2 production by resting neutrophils. Notably, when the initial level of LC-CL is high, additional effect of CO is negligible.
Our results taken as a whole allow to suggest the following hypothesis
on the role of EES in the functional interactions of white and red cells
in non-diluted blood. WBC produce ROS even in the absence of inflammatory
agents, and ROS reactions generate EES. Energy released due to their relaxation
to ground states is used for hemoglobin excitation and Hb-O2
dissociation. However, RBC release more O2 than WBC may consume,
and the major part of it goes into tissues. It is known that heme oxygenase
activity is increased under the conditions of tissue hypoxia. Thus, CO
may serve as an additional stimulus for O2 release that may
further increase WBC respiratory activity and EES generation. That is why
activity of these cells is high in venous blood especially under the conditions
of tissue hypoxia. However, intense generation of EES provides for sufficient
excitation of CO-hemoglobin and the release of CO from blood in the lungs.
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