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In vitro experiments with excessive and normal oxygenation of the culture medium showed unchanged oxygen consumption by mouse bone marrow cells under the influence of radioprotector indralin belonging to [alpha]1-adrenomimetics (100 [mu]g/ml). After exhaustion of oxygen in the medium below 10 [mu]M and progressive decrease in cellular respiration, indralin stimulated oxygen consumption by bone marrow cells by 1.5 times. The role of the observed effect of indralin in the realization of its radioprotective properties is discussed.[PUBLICATION ABSTRACT]
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Bulletin of Experimental Biology and Medicine, Vol. 155, No. 3, July, 2013 PHARMACOLOGY AND TOXICOLOGY
Effect of 1-Adrenomimetic Indralin on Oxygen
Consumption by Bone Marrow Cells in Vitro
M. V. Vasin, I. B. Ushakov, E. P. Korovkina, and V. Iu. Kovtun
Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 155, No. 3, pp. 337-339, March, 2013 Original article submitted February 1, 2012
In vitro experiments with excessive and normal oxygenation of the culture medium showed unchanged oxygen consumption by mouse bone marrow cells under the inuence of radio-protector indralin belonging to 1-adrenomimetics (100 g/ml). After exhaustion of oxygen in the medium below 10 M and progressive decrease in cellular respiration, indralin stimulated oxygen consumption by bone marrow cells by 1.5 times. The role of the observed effect of indralin in the realization of its radioprotective properties is discussed.
Key Words: 1-adrenomimetics; radioprotector; indralin; bone marrow; oxygen consumption by cells in vitro
The adaptive and trophic role of the sympathetic nervous system consists in maintenance of body system functions under conditions of their exhaustion via increasing tissue respiration and ATP synthesis. Catecholamines were found to stimulate oxygen consumption in the liver, kidneys, and muscles [8-11,14]. The effects of catecholamines on the hemopoietic system were never studied. At the same time, sympathetic innervation of the bone marrow was reported [12] and 1-, 2-, and 2-adrenergic receptors were found on hemopoietic stem cells and multipotent precursors (CD34+ and CD117+) [13].
Hyperactivation of adrenergic regulation under extreme conditions inevitably increases oxygen consumption with possible development of tissue hypoxia. In radiosensitive tissues, hypoxia is an important factor improving their radioresistance and largely determines the mechanism of action of radioprotectors, derivatives of biogenic amines [1]. Epinephrine, norepinephrine, and other sympathetic system agonists (mesaton and radioprotector indralin) exhibit potent radioprotective properties. However, circulatory hypoxia associated with increased peripheral resistance
and impaired blood ow in radiosensitive tissues is insufcient for manifestation of their radioprotective effect [1-3,6]. A hypothesis was put forward that tissue hypoxia plays a role in radioprotective action of these compounds [3].
The mechanism of the antiradiation action of Russian-made radioprotector of emergency action indralin (B-190 preparate) [5] is based on the direct 1-adrenomimetic effect [1,4]. Here we studied the effects of indralin on oxygen consumption by bone marrow cells in vitro for evaluation of the potential role of this factor in pronounced radioprotective properties of this compound.
MATERIALS AND METHODS
The study was conducted in accordance with Rules of the work using experimental animals (Order of the Ministry of Health Case of the Russian Federation, No. 267, June 19, 2003).
The work was carried out on outbred albino female mice (n=100) weighing 25-28 g. After decapitation, the bone marrow washed out of the femurs with 199 medium (0.15 ml/bone) using a syringe and dispersed by passing it repeatedly through a syringe needle.
A suspension of bone marrow obtained from three mice (6 bones) was pooled and placed into
Institute of Biomedical Problems, Russian Academy of Sciences, Moscow, Russia. Address for correspondence: mikhail-v-vasin@ yandex.ru. M. V. Vasin
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M. V. Vasin, I. B. Ushakov, et al.
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TABLE 1. Effects of Indralin on Oxygen Consumption by Mouse Bone Marrow Cells during Incubation of Cells in Medium 199
O2 concentration in medium 199 during incubation of bone marrow cells,
Change in oxygen consumption by bone marrow cells, % of initial level
control group(7 experiments)
1-ml thermostated plastic cell (37.5-38.5oC). The temperature was measured through a channel in the cell with a thermocouple of an electronic thermometer before, during, and after the experimental procedures.
Oxygen consumption by bone marrow cells was evaluated via polarographic measurement of oxygen level in the cultural medium without oxygen access. Platinum Clark electrode wrapped with polypropylene lm was used at a voltage of 0.6 V and a current of 7 A [7]. The current was recorded with a Minograph potentiometer at tape rate of 2 cm/sec.
After placing the measuring surface of the Clark electrode into the thermostatic cell, air bubbles were forced out by introduction of an additional amount of 199 medium through special channels in the thermostatic cell. After temperature and current stabilization, the bone marrow cell suspension was injected. After measurements, the number of bone marrow cells in the thermostatic cell was counted in a Goryaev chamber, it was equal to 150-200 million cells.
Indralin (1% solution in 199 medium) in a volume of 0.01 ml (100 g indralin) was added through a channel in the thermostatic cell. This concentration of the drug corresponded to optimum radioprotective dose of indralin (100 mg/kg) obtained in experiments on mice.
The rate of oxygen consumption by bone marrow cells in vitro was calculated after stabilization of pO2 reduction rate in the medium by measuring the gradient O2 level in the medium over 1-3 minutes and expressed in M O21012/Nmin per cell, where N is bone marrow cell count in the thermostated cell. Indralin was added to the thermostated cell after 1.5-fold drop of pO2 (~100 M O2).
The obtained data were statistically processed using Statgraphics Plus 3 software. The relationship between the rate of oxygen consumption by bone marrow cells and oxygen concentration in the medium was studied by correlation and regression analysis. The signicance of differences between the experimental and control groups was evaluated by nonparametric MannWhitney U test.
RESULTS
The initial oxygen concentration in the medium under conditions of medium temperature uctuation was ~192-198 M. The rate of O2 consumption by bone marrow cells in vitro in the rst half of the curve (during O2 concentration decrease to 100 M) remained practically unchanged and was ~157.88.4 M1012/
Nmin. Further decrease in pO2 was accompanied by progressive decline of oxygen consumption by bone marrow cells; at O2 concentration of 20 , the cell respiration decreased by 1.5 time (Table 1).
The curve describing the decline of oxygen consumption by mouse bone marrow cells during the decrease in oxygen content in the medium is satisfactorily approximated by an equation of the power function (Table 2). Under conditions of severe hypoxia (5 M 2 and lower), oxygen consumption by cells decreased sharply (by 2-5 times) until complete exhaustion of oxygen dissolved in the medium (Table 1).
In the presence of indralin (100 g/ml), the dependence of oxygen consumption by cells on oxygen level was also described by a power function. The curvature
indralin, 100 g/ml (6 experiments)
50.0-100.0 82.8 86.5
20.0-50.0 71.7 76.4
10.0-20.0 66.5 71.6
5.0-10.0 49.4 67.6*
0-5.0 19.2 29.3*
Note. *P<0.05 in comparison with the control group.
TABLE 2. Regression Equations of the Power Function y=axb Describing Indralin Effect on the Intensity of Cell Respiration with Decreasing O2 Content in the Medium
Group
Equation
my
Coefficient b
Correlation coefficient r
Indralin =44.50.183* 1.20 0.18300.0138* 0.92 (p<0.001)
Controls =31.10.263 1.39 0.2630.025 0.88 (p<0.001)
Note. y: decrease in O2 consumption by bone marrow cells (% of initial level); x: O2 level in the medium (M). *P<0.05 in comparison with the control.
Bulletin of Experimental Biology and Medicine, Vol. 155, No. 3, July, 2013 PHARMACOLOGY AND TOXICOLOGY
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of this curve estimated from coefcient b in the equation was signicantly higher than in the absence of indralin. Introduction of indralin at the stage of oxygen depletion in the medium (lower than 10 M) increased oxygen consumption by bone marrow cells 1.5 times (P<0.05; Table 1).
Thus, normal and hyperoxygenation of the medium, indralin had no effect on oxygen consumption by bone marrow cells under conditions of normal oxygenation and hyperoxygenation of the medium, but stimulated cell respiration under conditions of oxygen deciency in the medium. The stimulatory effect of 1-adrenomimetic indralin on cell respiration was ob-served at oxygen level in the medium <10 M, which was insufcient for the functioning of the hemopoietic system. This case can be an example of how the adrenergic system manifests its trophic function according the theory of L. A. Orbeli.
Stimulation of oxygen consumption by bone marrow cells in the presence of indralin under conditions of hypoxia can aggravate cell hypoxemia. Hence, indralin promoting circulatory hypoxia [1,2] and stimulating cellular respiration may increase cell hypoxemia in the bone marrow by 1.5 times. This feature of 1-adrenomimetic may contribute to the manifestation of its high antiradiation effect in large animals, incontrast to radioprotector mexamine (a derivative of serotonin) that has no effect on cell respiration despite its pronounced vasoconstrictor effect (not less pronounced than in indralin) [1-3,9].
The authors are grateful to Prof. L. M. Rozhdestvenskiy for helpful discussions.
REFERENCES
1. M. V. Vasin, Radioprotective Drugs [in Russian], oscow (2010).2. M. V. Vasin, V. V. Antipov, G. A Chernov, et al., Radiats. Biol. Radioekol., 37, No. 1, 46-55 (1997).
3. M. V. Vasin, I. B. Ushakov, L. V. Koroleva, and V. V. Antipov, Radiats. Biol. Radioekol., 39, Nos. 2-3, 238-248 (1999).
4. M. V. Vasin, I. B. Ushakov, L. A. Semenova, and V. Iu. Kovtun, Radiats. Biol. Radioekol., 41, No. 3, 307-309 (2001).
5. L. A. Ilyin, N. M. Rudny, N. N. Suvorov, et al., Radioprotector of Emergency Action Indralin. Radioprotective Properties, Pharmacology, Mechanism of Action, and Clinical Application [in Russian], oscow (1994).
6. V. I. Kulinskiy and L. I. Zolochevskaya, Radiobiologiya, 13, No. 3, 373-376 (1973).
7. L. M. Rozhdestvenskiy, S. S. Voronina, O. L. Smoryzanova, et al., Med. Radiol., No. 12, 38-43 (1984).
8. L. Breton, J. P. Clot, J. Bouriannes, and M. Baudry, . R. Seances Soc. Biol. Fil., 181, No. 3, 242-248 (1987).
9. K. A. Dora, S. M. Richards, S. Rattigan, et al., Am. J. Physiol., 262, No. 3, Pt. 2, H698-H703 (1992).
10. F. A. Gesek and J. W. Strandhoy, J. Pharmacol. Exp. Ther.,
249, No. 2, 529-534 (1989).
11. J. L. Hall, J. M. Ye, M. G. Clark, and E. Q. Colquhoun, Am. J. Physiol., 272, No. 2, Pt. 5, 2146-2153 (1997).12. G. J. Maestroni, Pharmacol. Res., 32, No. 5, 249-253 (1995).13. K. Muthu, S. Iyer, L. K. He, et al., J. Neuroimmunol., 186, Nos. 1-2, 27-36 (2007).
14. J. M. Ye, M. G. Clark, and E. Q. Colquhoun, Am. J. Physiol.,
269, No. 5, Pt. 1, E960-E968 (1995).
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