Ammonium Salts Promote Adaptive Changes of Rat Immune System to Ultimate Load in the Forced Swimming Model.

We studied the effect of different doses of ammonium chloride (ACl) and ammonium carbonate (ACr) on immunological parameters of the peripheral blood in rats during high-intensity exercise. Changes in the absolute and relative numbers of granulocytes, lymphocytes, natural killers, naive and mature effector cells one day after the end of the forced swimming cycle were found by using a hematological analyzer and a flow cytometer. Immunological indicators were analyzed relative to swimming duration on the last day of ultimate load. The revealed changes indicate the onset of the effector phase of the development of the inflammatory processes in the positive control group (physiological saline) and in rats receiving a higher dose of ACr (20 mg/kg), while administration of ACl prevented the development of inflammatory processes and shifts in the physiological balance of lymphocyte subpopulations. Immunological profiling indicates that ACl in a dose of 20 mg/kg most effectively improved physical performance in our forced swimming model.

Ammonium Salts Increase Physical Performance and Reduce Blood Lactate Level in Rats in a Model of Forced Swimming.

We compared the effects of two doses of ammonium chloride and ammonium carbonate (10 and 20 mg/kg) on the duration of swimming and blood lactate level. Ammonium chloride in a dose of 20 mg/kg was more efficient than in a dose of 10 mg/kg. The efficiency of ammonium carbonate in a dose of 10 mg/kg was similar to that of ammonium chloride in a dose of 20 mg/kg. Increasing the dose of ammonium carbonate to 20 mg/kg led to a decrease in the duration of swimming. On the last day of the experiment, lactate level in 5 min after exhausting load was maximum in control rats, while in rats treated with 10 mg/kg ammonium carbonate and 20 mg/kg ammonium chloride it was lower by 27 and 33%, respectively. In the control group, the amplitude of the decrease in lactate concentration in 1 h after load was 2-fold greater than in the group receiving ammonium chloride in a dose of 20 mg/kg and 1.6-fold greater that in groups treated with ammonium carbonate in a dose of 10 mg/kg and ammonium chloride in a dose of 20 mg/kg.

Ammonium Salts Promote Functional Adaptation of Rat Erythrocytes on the Model of Forced Swimming.

Ammonium, an end-product of catabolism, in low doses can promote adaptation of metabolic pathways in erythrocytes under conditions of extreme physical exercise. We compared the effects of two ammonium salts, ammonium chloride and ammonium carbonate, in two doses on biochemical parameters of rat erythrocytes 1 day after extreme physical exercise in a 4-week cycle of forced swimming. Of 16 analyzed parameters, the maximum number of significant shifts from the control was revealed in the groups of rats receiving ammonium chloride in doses of 20 and 10 mg/kg, and the minimal number of differences was found in groups treated with ammonium carbonate in the same doses. The comparison of the levels of reduced glutathione and 2.3-bisphosphoglicerate and activities of 5'-nucleotidase and Ca2+- and Na/K-ATPases attested to more rigorous control of the mechanism of oxygen delivery to tissues by erythrocytes after administration of ammonium chloride in a dose of 20 mg/kg.

Hemoglobin deoxygenation and methemoglobinemia prevent regulatory volume decrease in crucian carp (Carassius carassius) red blood cells.

Fish red blood cells (RBCs) exhibit an oxygen-dependent regulatory volume decrease (RVD) in hypoosmotic environment. In higher vertebrates, membrane-associated hemoglobin is involved in the regulation of osmotic ion movements across the cellular membrane. However, whether the hemoglobin conformational state plays a role in the regulation of osmotic responses in fish red blood cells is still not fully understood. We found that changes in hemoglobin conformation influence the pattern of the regulatory volume decrease in Carassius carassius red blood cells. In oxygenated cells (96.4 ± 3.7% oxygenated hemoglobin), the volume recovery was completed within 125 min. Deoxygenation of hemoglobin (96.5 ± 2.7% of deoxygenated hemoglobin) inhibited the volume decrease in hyposmotically swollen red blood cells. Reoxygenation restored regulatory volume decrease in cells within 5 min. Induced methemoglobinemia (48.4 ± 1.8% of methemoglobin and 41.3 ± 2.3% of deoxygenated hemoglobin) blocked the process of volume recovery and significantly decreased osmotic stability of red blood cells.

[The modification of method of erythrograms in patients under chronic hemodyalisis using hardware-controlled administration of laser low-angle light scattering].

The detailed analysis of structural characteristics of erythrocytes can be implemented with method of erythrograms. However its practical application in conditions of medical laboratory is a long and labor-intensive process of little avail for express-diagnostic. To register alterations of morphologic functional characteristics of erythrocytes in patients under chronic dialysis as compared with patients without renal pathology the new technique of low-angle light scattering never applied before for this purpose. Therefore, the purpose of this study is to resolve issue concerning validity of application of this technique for registration of alterations of functional status of erythrocytes in patients of department of chronic dialysis as compared with patients without renal pathology. The experiments concerning the identification of resistance of erythrocytes established significant differences for acid and ammonium models of lysis between patients without renal pathology and patients under chronic dialysis and also in patients in the course of dialysis session. In case of ammonium lysis, the differences were statistically significant between patients without renal pathology and patients under chronic hemodyalisis. In case of acid model, the differences were statistically significant in patients in course of dialysis session. Therefore, the application of low-angle light scattering technique is valid and informative for evaluation of functional status of erythrocytes in patients with terminal stage of chronic renal disease receiving treatment of regular hemodyalisis. The technique itself is low-cost, simple in application and easily reproduced. Therefore, the technique of low-angle light scattering can be applied both in research studies and clinical practice to identify characteristics of stability of membrane systems.

[Characterisation of enucleated cells apoptosis: human platelets and erythrocytes].

Apoptosis is a common mechanism of programmed cell death in virtually all nucleated cells. In spite of the fact that platelets and erythrocytes are the only enucleated cells in mammals they contain most of the apoptosis machinery of other cells and undergo similar apoptotic processes as nucleated cells except those connected with nuclear and chromatin transformation. Here we compare the mechanisms of platelet and erythrocytes apoptosis induced by different stimuli namely, stimulation ofthrombin and collagen receptor (T/C), inhibitor of BclX family proteins (ABT-373) for platelets, tert-butylhydroperoxide (tBH) and calcium ionophore (A-23187) for erythrocytes. Induction of platelet apoptosis by both methods (T/C and ABT-373) lead to strong phosphoetydilserine (PS) externalization, loss of the mitochondrial membrane potential (DeltaPsim), proteolytic cleavage of some cytoskeletal and regulatory proteins, and microparticle (MP) formation. However, there are clear differences between mechanisms of platelet apoptosis induced by TC and ABT-373. T/C induced apoptotic reaction is very fast (reach the maximum at 5 min), whereas ABT-373 induced reaction is more prolonged (first apoptotic evidence appears only after 30 min and reach the maximum after 3 hours). MP formation is much more pronounced in T/C than in ABT-373 stimulated platelets, whereas caspase 3 activation is much more stronger in ABT-373 than in T/C stimulated platelets. The main differences between these two apoptotic pathways are connected with aIIbp3 integrin, which activation appears only after T/C stimulation. For tBH experiments on erythrocytes we established optimal conditions (0.25x1012 cells/L, and strong, 1500 RPM stirring) for elucidation of apoptotic processes and found two independent ways of erythrocytes apoptotic processes; calcium independent, connected with met hemoglobin (metHb) formation (tBH stimulation), and calcium dependent pathway (A-23187 stimulation). Erythrocytes apoptosis induced by tBH is characterized by formation ofmetHb, cell shrinkage, fast (95 % during 3 hours) PS externalization, yield of hemoglobin, probably by vesicle (MP) formation. These cells are transformed to stomatocytes, become highly rigid, and could not be lysed even in pure water. All these reactions are calcium independent. Whereas increase of intracellular calcium concentration by A-23187 connected with formation of exinocytes, less pronounced (17 % during 3 h, 35 % during 15 h) PS externalization and rigidity (lysed in 50 mOsm buffer).

[Studies of interaction of intracellular signalling and metabolic pathways under inhibition of mitochondrial aconitase with fluoroacetate].

Mitochondrial aconitase has been shown to be inactivated by a spectrum of substances or critical states. Fluoroacetate (FA) is the most known toxic agent inhibiting aconitase. The biochemistry of toxic action of FA is rather well understood, though no effective therapy has been proposed for the past six decades. In order to reveal novel approaches for possible antidotes to be developed, experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells and cardiomyocytes, exposed to FA or fluorocitrate in vitro. The effect of FA developed at much higher concentrations in comparison with fluorocitrate and was dependent upon respiratory substrates in experiments with mitochondria: with pyruvate, FA induced a slow oxidation and/or leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides was prevented by incubation of mitochondria with cyclosporin A. Studies of the pyridine nucleotides level and calcium response generated in Ehrlich ascite tumor cells under activation with ATP also revealed a loss of pyridine nucleotides from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H under exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism, could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes. Highlighting the reciprocal relationship between intracellular pyridine nucleotides and calcium balance, we discuss metabolic pathway modulation in the context of probable development of an effective therapy for FA poisoning and other inhibitors of aconitase.

Cooperative type of platelet hypersensitivity to ADP.

We found that gestosis is associated with platelet hypersensitivity to ADP. Cell P2X1 receptors exhibited a positive cooperative response to ADP (EC(50)=10.88+/-3.70 nM, Hill constant n=2.59+/-0.50 rel. units). Cooperative binding of ADP to platelet P2X1 receptors was also observed during incubation of cells from pregnant women with isosorbide dinitrate.

Effects of oxytocin and prostaglandin F(2alpha) (enzaprost) on platelet aggregation.

The effects of uterotonic agents (oxytocin and enzaprost) on platelet aggregation in pregnant and nonpregnant women were studied by low-angle light scattering. In nonpregnant women oxytocin produced different effects on ADP-induced platelet aggregation: potentiation at low (<200 nM) and inhibition at high (>400 nM) ADP concentrations. In pregnant women oxytocin did not modulate ADP-induced platelet aggregation or this effect was negligible. Enzaprost competitively inhibited ADP-induced platelet aggregation in all examined women (inhibition constant 84.8+/-25.7 nM).

[Efficacy of purine nucleotides on purinergic P2 platelet receptors by small angle light scattering]. / Otsenka éffektivnosti deistviia purinovykh nukleotidov na P2-retseptory trombotsitov metodom malouglovogo svetorasseianiia.

The effect of purines on the activation and aggregation of thrombocytes in rats and rabbits was studied by the method of small-angle light scattering. The EC50 values of ADP, inducing the activation and aggregation of thrombocytes, reflect the sequence of the agonist action on various receptors: P2X1, 20-40 nM; P2Y1, 90-110 nM; P2YADP, 120-240 nM. It was demonstrated that ADP behaves as partial agonist not only with respect to P2X1 receptors, but with respect to P2Y1 receptors as well. Thrombocytes activated by 20 nM ADP or 100-nM ATP pass into a refracter state in the absence of further stimulation. The reaction halftime is tau 1/2 = 6.0 +/- 0.2 min for the cells activated with ADP and tau 1/2 = 16.5 +/- 0.2 min for ADP.

[Functional activity of blood platelets during cerebral ischemia/reperfusion in rats]. / Funktsional'naia aktivnost' trombotsitov pri ishemii/reperfuzii mozga krys.

In rats with an incomplete brain ischemia and subsequent reperfusion, the platelets sensitivity to the ADP decreased more obviously during the postischemic reperfusion. The platelets seem to be transformed into a refraction state after their activation in brain ischemia. The platelets refraction state might depend on a secondary activation of the nitric oxide-synthase in the platelets.

[The effect of a toxic dose of ammonium chloride on brain bioelectrical activity in rats]. / Vliianie toksicheskoi dozy khloristogo ammoniia na bioélektricheskuiu aktivnost' mozga krys.

A toxic dose of ammonium chloride (> 12 mmol/kg) caused death of animals within 10 min of i.p. injection, while pentobarbital--(40 mg/kg, i.p.) and kurare--(0.2 mg/kg, i.v.) injected rats died only in 11% of the tests. At 40 min after the injection of NH4Cl, the kurare-treated animals had minimum EEG amplitude in the cortex, maximum--in RF, and unchanged in amygdala. At the same time evoked potentials (EP) in RF induced by the periphery stimuli remained unchanged in form and increased in amplitude. Corticofugal impulses had no specific influence on the formation of EP in RF. The initial potentials were restored within 3 hrs. Thus, it could be concluded that ammonium differentiatively changed the state of the normal brain functional systems; it increased the afferentation and excitation of RF which could lead to violation of the vitally important functions especially breathing and cause the lethal outcome.