THE PECULIARITIES OF ARGINASE PATHWAY OF L-ARGININE IN SPERMATOZOA IN MEN WITH DIFFERENT FORMS OF PATHOSPERMIA.

The changes in arginase activity of spermatozoa and hormonal profile of peripheral blood of infertile men with various forms pathospermia have been studied. It has been found that arginase activity in the sperm cells of men with oligozoo-, antenozoo-, oligoastenozoo- and leucocytospermia is decreased in 2.1, 2.3, 2.4 and 3.3 times respectively. This indicates about inhibition of arginase pathway of L-arginine metabolism, which is not significantly dependent on the type of disruption of spermatogenesis. The most significant changes have been observed in infertile men with leucocytospermia since white blood cells stimulate the formation of reactive oxygen species, induction and development of oxidative and nitrative stress in spermatozoa. Inhibition of arginase pathway of L-arginine metabolism has adaptive role, which is to limit bioavailabil- ity of L-arginine and to prevent excessive formation of NO in cytotoxic concentrations to sperm cells. It has been noted changes in serum concentrations of gonadotropin and sex hormones in men with various forms of pathospermia. The most expressed significant changes were in levels of follicle stimulating hormone and testosterone. The concentration of follicle stimulating hormone in patients with oligozoospermia caused by hypogonadism is twice higher and in patients with leucocytospermia in 1.8 times higher than in fertile men. In patients with astenozoospermia this value is in 2.2 times lower than in normozoospermic samples but within the physiological norm. The testosterone level in men with oligozoospermia is in 1.6 times lower than in fertile men but within the physiological norm. It has been found that arginase inhibition of spermatozoa po6itively correlated with a decrease in their concentration in the ejaculate of infertile men with oligozoospermia (r =0.68).

[SPECIES COMPOSITION OF INFECTIOUS FACTORS THAT CAUSE THE REACTIVE ARTHRITIS DEVELOPMENT AND THEIR EFFECT ON ARGINASE-NO-SYNTHASE REGULATORY SYSTEM OF PERIPHERAL BLOOD LYMPHOCYTES].

The own observations results of urogenital, gastrointestinal and nasopharyngeal infectious factors that cause the development of reactive arthritis (PeA) are being presented. The greatest contribution to the development of this disease make Chlamidia trachomatis (36%), Streptococcus haemolyticus (pyogenes) (19%) and hepatitis viruses B and C (10%). As a result of the research a number of kinetic parameters of arginase and NO-synthase reactions in peripheral blood lymphocytes of patients with reactive arthritis was identified. The authentic increase of arginase activity in 3.3 times and eNO-synthase activity decrease by 1,9 times in peripheral blood lymphocytes of patients with PeA, compared to practically healthy donors were determined. Increased activity of arginase and iNO-synthase of lymphocytes indicates changes in immune cells functional activity, which may be due to impaired metabolic and regulatory processes in these cells caused by a bacterial or viral infection.

[Markers and regulatory mechanisms in ovarian carcinoma].

Ovarian carcinoma is one of widely spread malignant diseases of female reproductive system. Mortality rate from it is much higher than from other female malignant diseases. During last 20 years the level of ovarian carcinoma in Ukraine and vast majority of other countries remains high manifesting no signs of decrease. This arouses interest of researchers to development of new methods of early diagnosis, therapeutic approach, prognostic criteria, especially biochemical ones, and means of prophylaxis of this pathology in medical scientific society. At present there are no specific diagnostic tests which would allow revealing the tumor on the initial stages of its development. In spite of vide arsenal of tumor markers, the only reliable test for ovarian carcinoma is determination of antigen CA-125. The results of basic modern research are discussed in this survey. They are aimed at finding out regulatory mechanisms connected with metabolism of L-arginine, activities of ATP-hydrolase systems and searching new markers of ovarian carcinoma. The main possible candidates for this role are determined.

[Arginase and NO-synthase pathways of L-arginine metabolism in peripheral blood lymphocytes of patients with ovarian cancer].

The peculiarities ofarginase and NO-synthase pathways of L-arginine metabolism in peripheral blood lymphocytes of patients with ovarian cancer were studied. It was shown that the development of cancer pathology is associated with an imbalance in the NO synthesis in blood lymphocytes. The reason for such imbalance is the activation of arginase and inducible isoform of NO-synthase (iNOS) and significant inhibition of its constitutive isoform. The analysis of the kinetic properties of NOS of blood lymphocytes of patients with ovarian cancer was carried out. It was shown that the affinity constant of iNOS affinity for L-arginine is 5.4-fold lower than for eNOS of blood lymphocytes of persons in the control group. The inhibition of eNOS occurs via non-competitive type and is related to the reduction of maximum reaction rate.

[Kinetic properties of Na+, K+ activated, Mg2+ -dependent ATP-hydrolysis of blood lymphocytes in patients with rheumatoid arthritis and ankylosing spondyloarthritis].

The comparative analysis of the kinetic properties of ouabain-sensitive Na+, K+ -ATPase activity of saponin-perforated blood lymphocytes of donors and patients with rheumatoid arthritis (RA) and ankylosing spondyloarthritis (AS) was carried out. When analyzing the alterations in hydrolase activity of the examined enzyme it was shown that in the blood lymphocytes of patients with RA and AS the primary active transport of Na+ and K+ ions is less intensive in comparison with practically healthy donors, but it is characterized by almost the same capacity as in donors. The affinity constant of Na+, K+ -ATPase for ATP in the blood lymphocytes in patients with RA and AS is greater 3.1 and 2.5 times, respectively, in comparison with healthy donor. It was found that in conditions of rheumatic pathology in immunocompetent cells the inhibition of Na+, K+ -ATPase activity is not related to the reduction of maximum reaction rate, but is related to the decrease of Na+, K+ -ATPase affinity to ATP. However, Mg2+ -binding center of Na+, K+ -ATPase in patients with RA and AS remains native. It was identified that the affinity constant of Na+, K+ -ATPase to Na+ ions in the blood lymphocytes of patients with RA and AS is 2.75 times lower than its value in healthy donors. Na+, K+ -ATPase of the blood lymphocytes of patients with RA and AS retains its native receptor properties and sensitivity to ouabain does not change.

[Characteristics of Ca2+, Mg(2+)-ATPases of peripheral blood lymphocytes of patients with rheumatic pathology].

The analysis of the kinetic properties of Ca2+, Mg(2+)-ATPase of saponin-perforated peripheral blood lymphocytes of donors and patients with rheumatoid arthritis and ankylosing spondylitis was carried out. When analyzing the alterations in hydrolase activity of Ca2+, Mg(2+)-ATPase it was shown that affinity of Ca2+, Mg(2+)-ATPase of plasma membrane and membranes of endoplasmic reticulum for ATP do not significantly differ. It was found that the inhibition of examined enzyme systems occurs by mixed type both due to the reduction of maximum reaction rate and to the decrease of Ca2+, Mg(2+)-ATPase affinity for ATP in conditions of rheumatic pathology in the immunocompetent cells. It was identified that Ca2+, Mg(2+)-ATPase had significantly lower affinity for Ca2+ in lymphocytes of persons with rheumatic disorders than in donors.

[Comparative study of calixarene effect on Na+, K+ -ATPase activity in plasma membrane of contractile and mobile cells].

The paper is devoted to comparative analysis of the influence of a new class of macrocyclic compounds - calixarens on enzymatic activity of two ATP-hydrolase systems localized in the plasmatic membrane of contractile (myocytes of the uterus) and mobile (spermatozoids) cells--Na+, K+ -ATPase and basal Mg2+ -ATPase. The experiments performed on plasmatic membrane suspensions of myometrium and spermatozoids treated with detergent the authors studied the influence of calixarens C-97, C-99, C-107 (identified by the codes), functionalized with fragments of alpha-hydroxyphosphonic, alpha-aminophosphonic and methylenbisphosphonic acids accordingly, on enzymatic activity. The results have shown that C-97 and C-107 calixarenphosphonic acids in 100 microM concentration (97-99%) inhibit Na+, K+ -ATPase activity in both cases almost completely. C-99 (100 microM) calixaren appeared to be less effective with regard of its influence on the enzymatic systems under study: in the case of plasmatic membranes of myometrium suspension the activity of Na+, K+ -ATPase was decreased by 84-88%, and in the case of spermatozoids suspension--just by 15-20% of the control. All the studied calixarens (for both objects) in the maximal concentration (100 microM) practically did not influence the activity of basal Mg2+ -ATP-ase. The calixarens inhibited the enzymatic activity of Na+, K+ -ATPase more effectively than ouabain: in the first case the value of apparent inhibition constant I(0,5) was 25-100 nM, and in the second case--20-100 microM. The inhibition influence of calixarens on Na+, K+ -ATPase activity is characterized by the phenomenon of negative cooperativity (Hill's coefficient nH <1); the influence of ouabain in the case of plasmatic membranes of myometrium suspension is also characterized by negative cooperativity (nH < 1), and in case of spermatozoids suspension--by positive cooperativity (nH >1). The above results show that the studied calixarens are effective inhibitors of Na+, K+ -ATPase plasmatic membrane of contractive and mobile cells (C-97, C-99, C-107 calixarens in case of myocytes of uterus, and C-97, C-107 calixarens in case of spermatozoids).

[Eosin Y-sensitive ATPase activity in men's spermatozoids as a biochemical test for oligozoospermia].

The experiments performed on preparations of spermatozoids of men of reproductive age (27-44-year old) studied the ATPase activity (sensitive to inhibited effects of eosine Y) in both normal and oligozoospermia conditions when treating cell suspension with detergents. The methodical approaches for testing the so-called "common" and eosin Y-sensitive ATP-hydrolase activities in spermatozoids were developed. Saponin, the optimal detergent for permeabilisation of their plasma membrane was chosen using laser-correlational spectroscopia method. Saponin perforates effectively the membranes of spermatozoids, decreasing the average hydrodynamical diameter of cells from 10-15 microm (the spermatozoids themselves) to 3-8 microm (treating cell suspension with 0.05% solution of saponin) and even to 2-3 microm (treating spermatozoids suspension with 0.5% solution of detergent). A non-specific inhibitor of ATP-hydrolase's systems, eosin Y, decreases effectively the ATP-hydrolase activity of intact spermatozoids up to 40%. The exact effect of eosin depends on composition of incubation medium. In the model of extracellular conditions (the optimal concentration of detergent is 0.05%), eosin Y-sensitive ATP-hydrolase's activity of spermatozoids in both normal and oligozoospermia cases is increased by 220-240% (at an average). If enzymatic reaction was performed during intracellular conditions modeling (the optimal concentration of saponin is 0.5%), the increase of eosin Y-sensitive ATPase activity (up to 350-400% in normal conditions, and only to 130-150% in oligozoospermia conditions) was detected. This specificity can be used as easy-to-use clinical test for such pathology of men's reproductive system. Eosin Y inhibited doze-dependently the common ATPase activity in spermatozoids in both normal and with studied pathology. In both cases, after linearization of curves of catalytic titration of ATPase activity with eosin Y in Hill's plot the two-phase dependency, of high and low affinities, was found (the average values of imaginary inhibition constant I(0,5) are 0.1 and 0.3-0.4 mM correspondingly). In both normal and oligozoospermia conditions, the high-affinity component has a positive cooperativity, while the low-affinity component is characterized by a negative cooperativity. The obtained results may be of both theoretical and practical value for further investigation of membrane mechanisms used in the support of ion homeostasis in men's spermatozoids and its violation in conditions under different pathological states. Besides, the results can be used as a theoretical basis for improvement of simple and accessible clinical biochemical methods used for testing such a pathology as oligozoospermia.

[System of Ca2+ transport in spermatozoids and biochemical mechanisms of capacitation and acrosomic reaction].

The paper systematizes modern ideas regarding the role of macroelements (including calcium ions) in physico-chemical, biochemical spermatozoid processes, that provides the preservation of their biological completeness and fertility. Information concerning ion content and ion distribution in semen of males of different age groups and in the ejaculate with different quality indicators is presented. The basic ion-transport systems that are identified in spermatozoid membranes of males and their role in sperm activation, cell active movement ability, maintenance of cell homeostasis etc. are discussed. General schemes of biochemical mechanisms of capacitation process and acrosomic reaction are proposed.

Phosphorylation of phosphatidylinositides during muscarinic acetylcholine receptor regulation of myocardium contraction.

While estimating the myocardium sarcolemma phosphatidylinositides phosphorylation the muscarime acetylcholine receptor agonist carbacholine (10(-7) M) was determined as capable to stimulate 32P incorporation into phosphatidylinositol-4-monophosphate (in 2.6 times) and phosphatidylinositol-4,5-bisphosphate (in 2.3 times) indicating to the activation of phosphatidylinosite kinase and phosphatidylinosite 4-kinase respectively. The phosphorylation reactions in general completely depend on the presence in the incubation medium of Mg2+ capable in 10 mkM concentration to increase 32P influx into phosphatidylinositol-4-monophosphate 8 times, and into phosphatidylinositol-4,5-bisphosphate 4 times. Carbacholine (10(-7) M) also activates phospholipase C which hydrolyses phosphatidylinositol-4,5-bisphosphate. The latter is substantiated by the increase (2.6 times) of the secondary messenger--inositol-1,4,5-trisphosphate formation.

[Activity of membrane-bound protein kinase C and ATPase in erythrocytes in diabetic angiopathy]. / Aktivnost' membranosviazannoi proteinkinazy C i ATFaz eritrotsitov pri diabeticheskoi angiopatii.

The activities of protein kinase C, total, Mg2 and Na+, K(+)-dependent ATPases in red cell membranes were compared in 46 patients with insulin independent, 30 ones with insulin dependent diabetes mellitus with various degrees of vascular disorders, and in 17 patients with atherosclerosis with the predominant involvement of the main vessels of the lower limbs. Diabetes mellitus and the progress of vascular disorders were associated with a more marked depression of protein kinase C, total and Na+, K(+)-dependent ATPase activities, this being particularly characteristic of the patients with insulin-independent diabetes and macrovascular disorders. Inhibited activities of protein kinase C and ATPases in red cell membranes in the course of diabetic vascular disorders progress evidence their contribution to the pathogenesis of diabetic angiopathy.

[A mechanism of passive transport of Ca2+ in muscle sarcoplasmic reticulum]. / Mekhanizm passivnogo transporta Ca2+ v sarkoplazmaticheskom retikulume myshts.

Basing on the data available in literature and authors' investigations the mechanism of local alkalization of the myoplasm by proton efflux attended by Ca2+ influx is mic reticulum and may be the main link in the process of electrochemical coupling in the skeletal and cardiac muscle cells. Experimental evidence for participation of Ca2(+)-ATPase in the passive transport of calcium through sarcoplasmic membrane is given.

[The effect of Ca2+-phospholipid dependent phosphorylation on passive calcium transport in the myocardial sarcolemma]. / Vliianie Ca2+-fosfolipidzavisimogo fosforilirovaniia na passivnyi transport kal'tsiia v sarkolemme miokarda.

Protein kinase C in vesicular preparations of the myocardium sarcolemma is shown to phosphorylate proteins with the molecular weight of 250, 140, 67, 58, 24 and 11 kD. The exogenic protein kinase C catalyzed phosphorylation of the sarcolemma preparations lowers the initial rate of the passive calcium transport from 0.56 down to 0.18 mmol per 1 mg second. Activation of endogenic protein kinase C by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate is also accompanied by phosphorylation of vesicular preparations of sarcolemma and by inhibition of the passive calcium transport. Polymyxin B, being an inhibitor of protein kinase C, suppresses the phosphorylation and thus prevents the inhibitory action of phosphorylation on the passive calcium transport.

[Ca2+-phospholipid-dependent phosphorylation of vesicular preparations of myocardial sarcolemma]. / Ca2+-fosfolipidzavisimoe fosforilirovanie vezikulirovannykh preparatov sarkolemmy miokarda.

A Ca2+-phospholipid-dependent protein kinase C was isolated from the soluble fraction of bovine brain, using hydrophobic chromatography on phenyl-Sepharose CL-4B and high performance liquid chromatography on a Mono Q column. The enzyme had a specific activity of 822 nmol 32P/mg protein/min with histone H1 as a substrate. Phosphorylation of pig myocardium sarcolemma protein substrates was stimulated by Ca2+ and phosphatidylserine; the optimal concentrations of these compounds were 10(-4) M and 200 micrograms/ml, respectively. The value of Km(app) for Ca2+ was 3.10(-6) M. An addition of exogenous dioleine increased the enzyme affinity for Ca2+ which led to a decrease of Ca2+ concentration necessary for the maximal activation to occur. The optimal concentration of ATP needed for sarcolemmal preparation phosphorylation was 0.3-0.4 mM, which seems to be due to the high activity of sarcolemmal ATPases. The proteins phosphorylated in sarcolemmal preparations were identified, using SDS polyacrylamide gel electrophoresis with subsequent autoradiography. The 250, 140, 67, 58, 25 and 11 kD proteins appeared to be phosphorylated in the greatest degree. Since in myocardial sarcolemma protein kinase C predominantly phosphorylates the same proteins as does the cAMP-dependent protein kinase, it was assumed that protein kinase C can also play a role in the regulation of Ca2+-transporting systems of sarcolemma.

[ATP-dependent transport of Ca2+ in myocardium sarcolemma vesicles and its activation by phorbol esters]. / ATP-zavisimyi transport Ca2+ vezikulirovannymi preparatami sarkolemmymiokarda i ego aktivatsiia forbolovymi éfirami.

Highly purified pig myocardium sarcolemma vesicles possess the Ca2+,Mg2+-ATPase activity (4.1 mumol Pi/mg protein/hour) and induce the ATP-dependent accumulation of 45Ca2+ (6.0 nmol/mg protein/min). This reaction is not stimulated by oxalate; Ca2+ are released from the vesicles by saponin and Na+ treatment, which suggests that Ca2+ transport against the concentration gradient is induced by myocardium sarcolemma vesicles and not by sarcoplasmic reticulum fragments. The phorbol ester possessing a biological activity of a growth-promoting factor and activating membrane-bound protein kinase C stimulates the Ca2+,Mg2+-ATPase activity and the ATP-dependent accumulation of Ca2+, whereas its counterpart devoid of biological activity does not influence Ca2+ transport. Polymixin B, a specific inhibitor of protein kinase C, prevents the activating effect of phorbol esters on Ca2+ accumulation inside the vesicles. It is suggested that the ATP-dependent transport of Ca2+ in myocardium sarcolemma is controlled by Ca2+-phospholipid-dependent phosphorylation catalyzed by protein kinase C.

[Relation between the passive transport of calcium into vesicles of the myocardial sarcolemma and membrane potential]. / Zavisimost' passivnogo transporta kal'tsiia v vezikuly sarkolemmy miokarda ot membrannogo potentsiala.

The effect of membrane potential on the passive 45Ca2+ uptake by cardial sarcolemmal vesicles was investigated. Membrane potentials were generated by the K+ gradient in the presence of valinomycin and were measured using fluorescent dye diS-C3-(5). It was shown that the 45Ca2+ influx into vesicles increased twice after membrane depolarization. Evaluation of the 45Ca2+ influx over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. Passive 45Ca2+ transport was inhibited by 1 mM Cd2+ and Co2+. It is suggested that the voltage-dependent Ca2+ influx into vesicles occurs through Ca2+-channels.

[Passive transport of Ca2+ in vesiculated preparations of myocardial sarcolemma]. / Passivnyi transport Ca2+ v vezikulirovannykh preparatakh sarkolemmy miokarda.

The highly purified vesicles of myocardial sarcolemma oriented outward mainly by the cytoplasmic side are used to show that Ca2+-calmodulin-dependent phosphorylation inhibits passive Ca2+-transport, while R24571, a blocking agent of calmodulin-dependent processes, removes this inhibitory effect. Passive Ca2+ transport is also inhibited by nicardipin with Ki (5 X 10(-8) M) and Mg2+. Tetrodotoxin and tetraethylammonium exert no effect on Ca2+-transport.

[Potential-dependent passive transport of calcium in myocardium sarcolemma vesicles]. / Potentsialzavisimyi passivnyi transport kal'tsiia v vezikulakh sarkolemmy miokarda.

The effect of membrane potential on passive Ca2+ transport in isolated cardiac sarcolemmal vesicles was investigated. The membrane potentials were induced by creating potassium gradients across the vesicular membranes in the presence of valinomycin. The fluorescence changes in the voltage-sensitive dye, dis-C3(5), were consistent with the induction of potassium equilibrium potentials. The rate of 45Ca2+ efflux from inside-out vesicles was considerably greater at 0 than at -80 or +55 mV; prepolarization of the membrane to +90 mV did not enhance the 45Ca2+ efflux upon subsequent depolarization. The voltage-dependent 45Ca2+ efflux increased with a rise in internal Ca2+ concentration and exhibited a saturation effect. Furthermore, evaluation of the rate of 45Ca2+ efflux over a wide range of membrane potentials produced a profile similar to that of current-voltage relationships for single calcium channels in isolated cardiomyocytes. It is concluded that the voltage-dependent Ca2+ efflux from the vesicles occurs via Ca2+-channels.