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Сельскохозяйственная биология, 2011, № 2, с. 63-67.

УДК 636.2:614.876:591.111.1:577.15

ADENYLATE CYCLASE ACTIVITY IN LYMPHOCYTES AND THROMBOCYTES OF CATTLE AFTER RADIOACTIVE IRRADIATION

T.S. Shevchenko, I.V. Konopleva

The authors investigated the adenylate cyclase activity in the lymphocytes and thrombocytes of cattle exposed to total external γ-radiation at a dose of 3.5 Gy. After radiation action the basal and prostaglandin E1 stimulated components of adenylate cyclase activity in lymphocytes were manyfold increased, but these components in thrombocytes were significantly reduced. Reduction was reported in the adenylate cyclase stimulation

Keywords: cattle, external γ-irradiation, lymphocytes, thrombocytes, camp system ade-nylatе cyclase, coefficient of adenylate cyclase stimulation.

 

Life activities in most cells of mammals are controlled by a universal metabolism regulation system (messengers) (1, 2). The main extracellular regulators of physiological processes are hormones, mediators, neurotransmitters, intracellular – Ca²⁺ and the system of cyclic adenosine monophosphate (cAMP) (1-3). CAMP-dependent hormonal regulation can be described as follows: hormonal signal is accepted by corresponding receptors in the cell cytoplasm and causes a short-time increase in content of the secondary messenger cAMP, which then initiates a cascade of reactions leading to changes in metabolic processes and formation of a specific cell response (1, 2).
The action of γ-radiation in mammals leads to changes in contents of catecholamines, corticosteroids, serotonin and other biologically active compounds in tissues and peripheral blood (4). The post-radiation violation of cAMP system characteristics has been established in some cell populations of laboratory animals (5, 6), sheep and horses (7-9). At the same time, there’s almost no data on cattle enzymes of cAMP system, while it’s not always possible to extrapolate the results obtained on other animals, owing to specific differences of ruminants from other mammals. Furthermore, there can be manifested individual reactions of cattle cellular systems in response to irradiation.
The purpose of this work was studying the activity of the key enzyme of cyclic adenosine monophosphate - adenylate cyclase in the most radiosensitive cells of peripheral blood (lymphocytes and platelets) of cattle subjected to external γ-irradiation.
Technique. Black-and-White cattle (29 animals) were kept in the vivarium of the All-Russia Research and Development Institute of Agricultural Radiology and Agroecology (Obninsk). The diet of the animals was balanced for basic nutrients according to standards of the All-Russia Research and Development Institute of Animal Husbandry (Moscow). The experimental group (17 animals) was subjected to the total exposure of external γ-radiation at the half-lethal dose (3,5 Gy) at the radiation power of 1 Gy/h provided by the device GUZh-24 (radiation source - ¹³⁷Cs with gamma-quantum energy of  0,67 MeV) (Russia). The degree and uniformity of exposure were controlled by the dosimeter VAJ-18 (Germany) with a spherical ionization chamber VAK-253 (Germany). Non-uniformity of gamma-field was less than ± 15%. The control were all animals prior to irradiation and a separate group of non-irradiated cattle (12 animals) during 30 days.
The blood was collected from the jugular vein before irradiation and on the 1st, 3rd, 5th, 7th, 10th, 15th and 30th days after the exposure. Anticoagulant – sodium citrate at a final concentration of 0,38%. Populations of platelets and lymphocytes were isolated using the method developed by the authors (10). Isolated cells were washed twice against the solution containing NaCl, KCl, K₂HPO₄, MgCl₂, glucose and N-2-(hydroxyethyl) piperazine-N’-2-ethanesulfonic acid (respectively 145; 5; 0,5; 1; 3 and 10 mM, pH 7,4). Cells in the resulting suspensions were counted in Goryaev’s chamber; viability was assessed by testing with trypan blue. Prior to enzymatic reaction, an aliquot of platelets was frozen,  an aliquot of lymphocytes was lysed in hypotonic medium for 30 minutes at 4 °C. Adenylate cyclase activity in cellular lysates was determined by the method described previously (8). The labeled substrates were [14C]-ATP and [3H]-cAMP (“Amersham”, UK). Radioactivity of the samples was measured using the liquid-scintillation counter SL-4220 (“Intertechnique”, France). The hormone-stimulated component of adenylate cyclase activity was determined as the difference between enzymatic activity of the activator (prostaglandin E1) and without it. Coefficient of stimulation of adenylate cyclase was calculated as the ratio of stimulated and basal components of activity.
Statistical processing of results was performed using Student's t-test in Microsoft Excel 2003. Differences of values were assumed to be significant at p < 0,05.
Results. The analyzed isolated populations of lymphocytes and platelets showed good viability (90-95% upon the results of trypan blue test).
In quiescent cells (without stimulatory or inhibitory effect of physiologically active substances), the functioning of adenylate cyclase is associated with catalytic subunit of the enzyme. This is a so-called basal component of the activity (1). In non-irradiated cattle lymphocytes it amounted to 2,1±0,4 pmol/min½106 cells, in platelets - 13,3±3,0 pmol/minx10⁸ cells. The level of enzyme activity remained almost unchanged for 30 days. In lymphocytes of irradiated animals, this parameter exceeded control on the 1st day – in 4,33 times, on the 3rd – in 3,76, on the 5th – in 4,29, on the 10th - at 3,57, on the 30th day – in 2,74 times (Table). Basal activity of adenylate cyclase in cattle platelets on the 1st day after the radiation exposure was reduced in 3,09 times compared with initial level. On the 3rd -5th days, there was recorded its elevation back to control values. On the 7th day, it decreased in 2,66, on the 10th – in 3,25, on the 15th – in 5,71 times (Table).
The mechanism of cAMP-dependent hormonal regulation of cellular metabolism is mediated by adrenergic receptors embedded in cytoplasmic membrane of the cell, G-protein and adenylate cyclase. When two first components are unchanged, the transduction of a hormonal signal is realized by increasing or inhibiting adenylate cyclase activity. In experiments on laboratory animals, the commonly used activators are b-isoproterenol or prostaglandin E affecting, respectively, b- and a-receptors of adenylate cyclase complex (11). Since isoproterenol activates adenylate cyclase in lymphocytes of laboratory animals and inhibits it in platelets, while prostaglandin E1 stimulates the enzyme in both cases, so the latter was selected to be the effector of cAMP-dependent hormonal regulation.
At presence of 10^-5 M prostaglandin E1, basal activity of adenylate cyclase in lymphocytes of non-irradiated cattle increased by 70-110, in platelets - by 100-400%. Stimulated component of adenylate cyclase activity provided by functioning of the hormone-binding enzyme subunit in lymphocytes and platelets were, respectively, 1,08±0,18 pmol/min x 10⁶ cells and 25,4±5,1 pmol/min x 10⁸ cells. On the 1st day after irradiation,  this component increased in the lymphocytes in 3,19 times, on the 3rd – in 3,54, on the 5th – in 6,56, on the 10th – in 2,97 and on the 30th – in 2,58  times compared with control (Table). In platelets, the prostaglandin-stimulated component of adenylate cyclase activity reduced on the 1st day in 1,82 times, on the 3rd day – in 4,75, on the 5th – in 1,74, on the 7th – in 4,89, on the 10th – in 10, 91 and on the 15th day – in 4,89 times.

In lymphocytes, coefficients of adenylate cyclase stimulation were almost similar in control and irradiated animals (Table). In platelets of the irradiated cattle, this parameter decreased on the 1st day in 2,04 times, on the 3rd – in 5,06, on the 5th – in 1,44, on the 7th – in 1,78 and on the 10th day – in 3,14 times.
The data obtained on the 15th-30th days were not reliable, as most of irradiated animals had died by this time.
The hormone-stimulated component of adenylate cyclase activity and coefficient of enzyme stimulation characterize the efficiency of hormonal signal transduction in cells (6). Therefore, it can be assumed that after the exposure of cattle to external γ-radiation, the efficiency of transmission of hormonal stimuli in lymphocytes was the same as in non-irradiated animals, or it became higher than in control (considering the rise of the hormone-stimulated component of adenylate cyclase activity). In platelets of irradiated animals, the reduced efficiency of transmission of the hormonal signal was indicated by inhibition of prostaglandin-stimulated component of adenylate cyclase activity and lower value of stimulation coefficient of the enzyme.
Thus, in both studied types of blood cell, there were established the changes in basal and hormone-stimulated components of adenylate cyclase activity, and in blood platelets there was recorded the decrease of stimulation coefficient. These facts suggest that both subunits of the enzyme (catalytic and regulatory) were affected by processes occurred in the enzyme complex. Functional activity of the enzyme primarily depends on the conformation of its macromolecule and its environment in the cytoplasmic membrane (6). Biological membranes are one of targets for ionizing radiation (12). The observed post-irradiation changes in adenylate cyclase activity can be caused by radiation-induced violation in structure and functioning of cytoplasmic membranes of studied cells.
At the same time, there was expected that post-radiation disturbances in cell membranes can change coefficients of stimulation of adenylate cyclase in both platelets and lymphocytes. In this experiment, such fact was found only in platelets. It’s possible, there were other factors influencing the activity of adenylate cyclase in studied cells. It is known that external γ-radiation affects subpopulation structure of lymphocytes in the peripheral blood of animals: there are massive deaths of most radiosensitive cells and increase in relative content of radioresistant cells of a particular population (13, 14). B-lymphocytes highly sensitive to radiation are a small fraction of all lymphocytes in the peripheral blood of animals (about 20% in laboratory animals, 15-33% - in cattle) (15, 16). This subpopulation of cells normally lives 1-5 days, and after 3 days of g-irradiation, the number of these cells in peripheral blood of animals is very small (16, 17).
T-lymphocytes are the more radioresistant and long-living cells (duration of life – 200-300 days) (16). So, there are mainly T-lymphocytes representing the population of lymphocytes circulating in the bloodstream after irradiation of animals. It can be assumed that the increased functional activity of adenylate cyclase in lymphocytes is associated not only with postradiation modification of cell membrane, but (to a greater degree) this fact can be the specific feature of cells remaining in the bloodstream after the death of most radiosensitive subpopulation of lymphocytes and the young lymphocytes coming into the blood from the bone marrow pool. Lymphocytes of the peripheral blood of irradiated animals have higher resistance to radiation and adenylate cyclase activity. Platelets are even more radioresistant than T-lymphocytes. Normally, they live in the bloodstream 5-10 days (18). By the 7th -10th day, subpopulation of platelets in the peripheral blood becomes significantly renewed. In the initial period of irradiation, the effect of g-radiation on adenylate cyclase activity of platelets reveals itself mainly as violations in the cell membrane, while the changes in enzyme activity on the 7th-10th day can be associated with new cells released from the bone marrow pool into the blood and having qualitatively different properties. The renewed platelets population has significantly reduced levels of adenylate cyclase activity (basal and stimulated by prostaglandin E1) and the coefficient of stimulation; therefore, there is less efficient transduction of the hormonal signal transmission in cells.
Thus, the exposure of cattle to external gamma-radiation contributed to change in activity of adenylate cyclase - the key enzyme of cyclic AMP system in peripheral blood cells. From the 1st to the 30th day of irradiation, in cattle lymphocytes there was recorded an increase of both basal and prostaglandin E1-stimulated components of enzyme activity. In platelets, from the 1st to the 15th day there was observed the suppression of both components of adenylate cyclase activity. The coefficient of stimulation of adenylate cyclase decreased in platelets and remained unchanged in lymphocytes. It can be assumed that the radiation-induced change in activity of adenylate cyclase in these cells was the result of two factors: violations of structural-functional state of the cytoplasmic membrane and the modified composition of cell subpopulation circulating in the peripheral blood. In the initial period of radiation exposure (from the 1st day), enzyme activity changes owing to impact on cell membranes. In later period, enzyme activity is determined by predominance in the peripheral blood of lymphocyte subpopulations with higher resistance to radiation injury and increased activity of adenylate cyclase and the renewed population of platelets having reduced activity of this enzyme.

 

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All-Russia Research and Development Institute of Agricultural Radiology and Agroecology, RAAS, Kaluga province, Obninsk 239032, Russia e-mail: riar@obninsk.org, Shevchenkotatyana@yandex.ru

Received December 22, 2008