Role of Na+,K+-ATPase in Bone Remodeling.

The effects of cardiotonic steroids (ouabain and digoxin) on the bone formation were studied using the organotypic tissue culture in combination with confocal microscopy. The expression of α1- and α3-isoforms of Na+,K+-ATPase was detected in cells of the bone tissue of 12-day-old chicken embryos. Ouabain in a concentrations 10-10 M (comparable with its endogenous concentration) can modulate transducer function of Na+,K+-ATPase and control the growth and proliferation bone tissue cells. Unlike ouabain, digoxin is not involved in the regulation of bone tissue growth in a 12-day-old chicken embryo.

[The role of polypeptide compounds in mechanism of CNS plasticity in patients with hereditary pathology of peripheral motor neuron]. / Rol' rostovykh polipeptidnykh soedinenii v mekhanizme plastichnosti TsNS u bol'nykh nasledstvennoi patologiei perifericheskogo dvigatel'nogo neirona.

Synaptic pruning is a physiological mechanism of neuroplasticity, which is regulated through synthesis of growth polypeptides, neurotrophins. The role of neurotrophins in the mechanism of synaptic pruning in patients with hereditary pathology of peripheral motor neuron was studied in a clinical experimental trial. It was found that patients had elevated levels of regulatory growth polypeptides, which led to the axon growth inhibition effect in organotypic tissue cultures. Thus, neurotrophin overexpression can be considered as a factor preventing synaptic pruning and contributing to further process of neurological degeneration in nerve tissue in patients with hereditary pathology of peripheral motor neuron.

[Remodeling of Cardiovascular System: Causes and Consequences].

Literature and our data suggest the regulatory action of a number of biologically active substances (catecholamines, cardiac glycosides, ß-blockers, angiotensin-converting-enzyme inhibitor) on the growth and proliferation of heart cells. By using of organotypic tissue culture has proved that the basis of this regulation is the ability of test substances, receptor- or transducer-mediated signaling to modulate the function of Na⁺, K⁺-ATPase. There is a delay in the development of vascular smooth muscle in the late postnatal period in rats with the blockade of the sympathetic nervous system in the prenatal period. The relationship between vascular remodeling and contractile activity is described. It seems that one of the causes of high blood pressure is a remodeling of the cardiovascular system, which precedes the development of hypertension.

[INVOLVEMENT OF р38 МАРК OF SENSORY NEURONS IN OUABAIN-ACTIVATED SIGNALING CASCADE].

Recent investigations of the new transducting function of Na+,K+-АTPase is of great interest. Na+,K+-АTPase might be a target (receptor) for cardiotonic steroids. This membrane protein switches on intracellular signaling mechanisms due to its ligand-receptor interactions with the steroids that, in turn, result in activation of intracellular cascade processes. р38 belongs to mito-gen-activated protein kinase family (MAPK). The kinase transduces signals of wide range intra-cellular signaling cascades that control proliferation, differentiation, and survival. Using embryonic organotypic tissue culture method the present work shows that р38 МАРК is involved in the signal transducting process in series with Na+,K+-АTPase.

[THE USING OF THE METHOD OF RECONSTRUCTION OF OPTICAL CROSS SECTIONS FOR INVESTIGATION OF THE TROPHOTROPIC EFFECTS OF ADRENALINE AND ATENOLOL].

The trophotropic effects of epinephrine and atenolol were investigated by using the organotypic culture method combined with the method of the reconstruction of optical cross sections. For the best possible image contrast was experimentally established the correction for Petri dish bottom thickness equal to 1 mm (objective lens Carl Zeiss). It was shown by method of the reconstruction of optical cross sections that growth zone in organotypic culture forms a multidimensional structure. In the presence of atenolol (10(-4) M) the thickness of the growth zone of the cardiac tissue explants was above the reference value 212%. The thickness of the growth zone in the presence of epinephrine (10(-9)-10(-13) M) did not differ from the control values. Experimentally was proved that atenolol mimics the trophotropic effects of epinephrine and these effects of epinephrine is realized primarily through ß1-adrenergic receptors.

The effect of marinobufagenin on the growth and proliferation of cells in the organotypic culture.

In recent years, a substantial interest has been aroused in investigating Na(+),K(+) ATPase as a membrane structure which not only performs its direct function in maintaining the gradients of Na(+) and K(+) concentrations, but also may participate in the intracellular signal transduction processes in response to various physiological stimuli. The effect of marinobufagenin, a digitalislike factor, on the growth and proliferation of sensory ganglia, cardiac, retina, skin, and liver tissue explants of 10-12day old chicken embryos was investigated by the organotypic culture method in a wide range of concentrations (10(-10) to 10(-4) M). It was first demonstrated that marinobufagenin inhibited the growth of the investigated tissue explants in the dosedependent but not tissuespecific manner. The experimental data obtained allow us to suggest that the mechanism of regulation of tissue growth by marinobufagenin during the embryonic period of ontogenesis is associated with the modulation of the pump function of Na(+),K(+) ATPase but not its transducing function.

[The influence of ouabagenin on the growth and proliferation of cells in the organotypical culture].

The aim of the present work was to investigate the effect of ouabagenin on the growth and proliferation of cells in organotypic culture. The objects of study were explants of nerve, cardiac, retina and liver tissue of 10-12 day old chicken embryos. Inhibitor of Na+,K(+)-ATPase ouabagenin was investigated in a wide range of concentrations (0.1 nM-1 mM). It has been found that the ouabagenin controls cell growth and proliferation in a dose-dependent manner and tissue-unspecific. The data obtained show that ouabagenin regulates only the pumping function of Na+,K(+)-ATPase.

Effect of ouabain on growth of skin explants in organotypic culture.

Organotypic culture was used to study the effects of glycoside ouabain and its aglycone ouaba-genin on the growth of skin explants from 10-12-day chicken embryos. The tested agents demonstrated dose-dependent inhibition of skin growth. The effective concentrations imply that ouabain and ouabagenin mediate their inhibitory effect on the skin growth via interaction with α1-isoform of Na(+),K(+)-ATPase that plays mostly the role of ionic pump.

Impact of norepinephrine and selective ß1-adrenoceptor blockers on the growth of retinal tissue explants.

We studied the effect of norepinephrine and selective ß(1)-adrenoceptor blockers on the growth of retinal tissue explants from 10-12-day-old chicken embryos in organotypic culture. The test drugs produced a dose-dependent effect on cell proliferation. Norepinephrine (10(-12) M) and atenolol (10(-4) M) are shown to significantly stimulate explant growth. Combined treatment with norepinephrine (10(-12) M) and atenolol (10(-4) M) results in cumulation of their stimulatory effects. The results suggest that the retina-stimulating effect of norepinephrine is based on its interactions with other types of adrenoceptors.

Effect of anoceptin on detoxifying function of the liver.

The absence of negative effects of anoceptin on the liver tissue and detoxifying function was proven experimentally in vitro and in vivo. It was proven for the first time that comenic acid (anoceptin active substance) in concentrations of 10(-4)-10(-12) does not modulate the growth of liver explants from 10-12-day chicken embryos. The effect of anoceptin on the detoxifying function of the liver was studied on Wistar rats under conditions of hexenal-induced sleep. The drug was injected into the caudal vein for 90 days in a daily dose of 5, 100, or 300 mg/kg. The duration of hexenal-induced sleep was evaluated before and on days 30 and 90 of the study. Anoceptin did not modify the duration of hexenal-induced sleep and the status and detoxifying function of the liver.

[Role of oubain in regulation of growth of different tissues].

Using the method of organotypical cell culture, experimental data was obtained supporting the hypothesis that chelate complex ouabain-Ca2+ modulates the transducer function of Na+, K+- ATPase. Quantum-chemical calculations helped to elucidate two principally distinct modes for chelation of Ca2+ ions by ouabain molecule. It is predicted that ligang-receptor complex of ouabain-Ca2+--Na+,K+-ATPase is formed due to ion-ionic bonds. The forming of this complex switches on the transducer function of the sodium pump. Our data also show that removal of free Ca2+ ions from the cell culture media using EGTA has no effect on binding of ouabain with pumping control site of Na+K+-ATPase.

The effects of sodium pump inhibitors on sensory ganglion neurite growth.

The effects of sodium pump inhibitors of the cardiac glycoside family (strophanthin K and digoxin) on neurite growth in the sensory ganglia of chick embryos (10-12 days) were studied in organotypic tissue cultures. These experiments produced the first evidence that these medicinal agents have marked neurite-suppressing actions. Their effects on sensory ganglion neurite growth were dose-dependent. At 1.10(-6) M, strophanthin K and digoxin completely blocked sensory ganglion neurite growth. Addition of study compounds to the medium at a concentration of 1.10(-7) M decreased the area index of the experimental explants to a level significantly below that of the controls, by a mean of 45%. These strophanthin K and digoxin concentrations were comparable with those at which endogenous digitalis-like factors are present in the systemic circulation. These results provide evidence that cardiac glycosides can produce the directed regulation of nervous tissue growth by affecting the signal transducer Na+, K+-ATPase.

[Effect of Na(+), K(+)-pump inhibitors on the sensory ganglia neurite growth].

Inhibitors of Na(+), K(+)-pump belonging to the class of cardiac glycosides were investigated in organotypic tissue culture of dorsal root ganglia cells of 10-12 days old chicken embryos. The data obtained show that the application of cardiac glycosides (strophantin K and digoxin) in a wide range of concentrations controls the neurite growth in sensory neurons in the dose-dependent manner. It was shown, that at the concentrations of cardiac glycoside exceeding 1 x 10(-6) M the growth of neurites was totally inhibited. Our data indicate that cardiac glycoside have the down-regulation effect on the neurite growth. The data obtained indicate that the Na(+), K(+)-ATPase is involved in the control of the process of neurite growth as a signal transducer.

Drug modulation of transductor function of Na+,K+-ATPase.

Experimental proof of the hypothesis on modulation of the transductor function of Na+,K+-ATPase by the ouabain-Ca2+ chelate complex was obtained by the method organotypic tissue culture. Quantum chemical estimations detected two principally different modes of Ca2+ ion chelation by ouabain molecule. It is hypothesized that the ouabain-Ca2+-Na+,K+-ATPase ligand-receptor complex is formed due to ion-ion bonds. The formation of the complex serves as the signal triggering the enzyme transductor function. It is experimentally proven that ouabain is incapable of inhibiting neurite growth in sensory neuron and heart tissue explants after removal of free calcium from the nutrient medium with EGTA.

Role of cardiac glycosides in regulation of the growth of retinal tissue explants.

We studied the role of cardiac glycosides in the regulation of the growth of retinal tissue explants from 10-12-day-old chicken embryos in organotypic culture. The studied compounds produced a dose-dependent effect on cell proliferation in retinal tissue explants. Ouabain (10(-13)M), strophanthin K (10(-13)M) and digoxin (10(-11)M) significantly stimulated explant growth. It was hypothesized that the physiological role of endogenous ouabain is associated with regulation of tissue modeling.

Effect of beta-adrenoceptor blockers on growth of neurites in spinal ganglia in organotypic tissue culture.

We studied the effects of norepinephrine and beta1-adrenoblockers atenolol and metoprolol on the growth of neuritis in organotypic tissue culture of spinal ganglia from 10-12-day chicken embryos. Atenolol (10(-4) M) accelerated the growth of neurites in spinal ganglia by 45%, while metoprolol inhibited the growth of the explants almost in all examined concentrations (10(-10)-10(-4) M). Thus, beta1-adrenoblockers can produce opposite effects on neurite growth. Both atenolol and metoprolol abolished the neurite-stimulating effect of norepinephrine, probably, because this effect is mediated via beta1-adrenoreceptors of spinal ganglia.

Regulatory role of Na,K-ATPase in the growth of heart tissue explants in organotypic culture.

We studied the effects of ouabain, norepinephrine, and acetylcholine on the growth of heart tissue explants from 10-12-old chicken embryos in an organotypic culture. Ouabain, norepinephrine, and acetylcholine stimulated the growth of explants. Our results suggest that trophic function of acetylcholine and norepinephrine is associated with regulation of Na,K-ATPase activity as a signal transducer.

Role of Na/K-ATPase in regulation of neurite growth in sensory neurons.

We studied the effects of acetylcholine and norepinephrine on the growth of neurites in organotypic culture of 10-12-day-old chick embryo sensory neurons. Acetylcholine (10(-8) M) and norepinephrine (10(-13) M) stimulated the growth on sensory neuron neurites. Experiments with combined application of acetylcholine and norepinephrine against the background of ouabain showed that the nonspecific action of the test neurotransmitters is related to modulation of Na/K-ATPase activity.

[Comparative analysis of the cardiac glycosides action on the growth of the cardiac tissue explants].

The method of organotypical cell culture was used. The long-term cell culture of cardiac embryonic tissue of 10-12-days old chicken was investigated. The effects of ouabaine, strophantin K and digoxin on the growth of cardiac tissue explant were measured. The ouabain concentration which stimulates activity of Na+, K+-ATPase as the signal transducer, was determined. It was equal to 10(-10) M. Strophantin K and digoxin stimulate growth of cardiac tissue in concentration equal to 10(-16) M and 10(-18) M, resp. The data obtained show that application of cardiac glycosides led to control of cardiac tissue growth in dose-dependent manner. We hypothesize that alpha3 isoform of Na+, K+-ATPase is a signal transducer that controls the cardiac cell metabolism and growth.