PROBABLE INVOLVEMENT OF REACTIVE OXYGEN SPECIES IN THE REGULATION OF THE OXYGEN DELIVERING FUNCTION OF BLOOD.
K.N. Novikov, V.L. Voeikov, N.D. Vilenskaya, O.A. Leontieva
Faculty of Biology, Lomonosov Moscow State University, Moscow
Free radical oxidative reactions generating electron excited species are continuously running in living organisms. Relaxation of electron excitation may be followed with low-level luminescence. A well known example of such processes is chemiluminescence (CL) that accompany the respiratory burst (RB) in neutrophils. RB in neutrophils is induced by alien particles, such as microbes, and is characterized by many-fold intensification of reactive species (ROS) generation.
Neutrophil CL is usually studied in the suspensions of isolated cells or in highly diluted blood. However, using highly sensitive photon detectors, we managed to demonstrate low-level luminescence of non-diluted human blood especially of blood of heart ishaemic disease patients. Photon emission intensity may be strongly amplified by addition of CL indicators such as luminol (LM – a probe for O2·¾, H2O2, OH·, OCl¾`), or lucigenin (LC – a specific probe for superoxide radical, O2·¾) to blood. The levels of LM-dependent CL (LM-CL) in fresh blood of healthy donors in the absence of neutrophil activators usually slightly increases after LM addition and than declines to background values. On the contrary, LC-CL intensity in such blood rapidly rises up to values many-fold exceeding that of background and does not decline for many hours. CL patterns in 8-fold and more diluted blood and in purified neutrophil suspensions are very different from those of non-diluted blood: LC-CL is very low, while LM-CL intensity after neutrophil stimulation is much higher than in non-diluted blood. CL pattern in non-diluted blood depends upon a particular donor – in some donors’ blood LM-CL levels may be close to that of. In cases of inflammation and in ischaemic disease patients’ blood LM-CL may even highly exceed LC-CL.
High intensity of LC-CL in fresh non-diluted healthy donors’ blood indicates that leukocytes, and in particular neutrophils continuously generate O2·¾. As more that 99% of oxygen in blood is bound to hemoglobin, leukocytes can get it only from erythrocytes. Interaction of white and red cells in blood may be under the control of various factors, it may depend upon blood state and may change under pathological conditions. Taking this into consideration we studied the effects of passive blood aeration vs anoxic conditions upon LC-CL and LM-CL. LC-CL levels and dynamics were the same in aerated blood and in blood isolated from the air. On the other hand, LM-CL begins to increase after addition to blood of neutrophil activators only in the aerated blood. However, when LM-CL intensity reaches some particular level, cessation of blood contact with the air does not ssignificantly change the time course of LM-CL development. Thus, blood contact to the air is needed presumably only for the process of RB initiation, while after its «flaming up» it may be produced due to oxygen supplied by erythrocytes. We suggested that the «air ions» — O2·¾ present in the ambient air, may play this priming role. The importance of ROS present in the air for the RB was demonstrated in the following experiment. 10-12 hours after RB initiation in the passively aerated blood LM-CL in it declines nearly to the background values. Transient and weak elevation of LM-CL may be induced by passing 1-2 ml of the air through «exhausted» blood. However, 10-15 min after blood had been bubbled with only 10-50 mkl of 02 obtained from 3% Н2О2 during its decomposition the new intense wave of LM-CL was generated that lasted for many hours. It is most probable that the traces of O2·¾ in fresh О2 are playing the triggering role for RB reinitiation.
LC-CL dependence upon oxygen supply from erythrocytes in non-diluted blood allowed us to observe a new phenomenon concerning physiological function of CO (carbon monoxide, coal gas) in oxygen supply to cells and tissues. It is known that in spite of high CO toxicity when it is present in the inhaled air this gas is produced in the organism as a normal product of the ubiquitous enzyme heme-oxygenase. CO as well as NO (nitrogen oxide) are considered to regulate the activity of the heme-containing enzyme, guanylate cyclase. The latter produces cyclic GMP – the regulator of blood vessel tonicity. However, this hypothesis overlooks that hemes are much more abundant in hemoglobin and that CO affinity to hemoglobin 220-fold exceeds that of O2. Thus CO generated by heme-oxygenase should in the first place stick to hemoglobin of erythrocytes. We have found that if several microliters of CO are passed through non-diluted blood a strong elevation of LC-CL is observed. On the contrary, when CO is passed through the suspension of isolated neutrophils both LC-CL and LM-CL intensity declines presumably due to blockade by CO of heme-containing enzymes responsible for ROS production. Sharp elevation of LC-CL after CO passage through blood indicates that this gas does not just binds to free hemoglobin molecules, but that each binding event triggers О2-heme dissociation in many other hemoglobin molecules.
In conclusion our results demonstrate that 1) neutrophils may have at least two different modes of ROS production revealed differentially by LC-CL and LM-CL; 2) neutrophils continuously generate ROS from oxygen supplied by erythrocytes in fresh non-diluted blood of healthy donors. We speculate also that endogenous СО stimulates the bursts of О2 liberation by erythrocytes, that is needed for operative oxygenation of tissues with oxygen, and, in particular, for the intensification of ROS production by neutrophils, that is mirrored by LC-CL intensity elevation.