[The involvement of actin cytoskeleton in glutoxim and molixan effect on intracellular Ca(2+)-concentration in macrophages].

Glutoxim and molixan belong to new generation of disulfide-containing drugs with immunomodulatory, hepatoprotective and hemopoetic effect on cells. Using Fura-2AM microfluorimetry, two structurally distinct actin filament disrupters, latrunculin B and cytochalasin D, and calyculin A, which causes actin filaments condensation under plasmalemma, we have shown the involvement of actin cytoskeleton in the intracellular Ca(2+)-concentration increase induced by glutoxim or molixan in rat peritoneal macrophages. Morphological data obtained with the use of rhodamine-phalloidine have demonstrated that glutoxim and molixan cause the actin cytoskeleton reorganization in rat peritoneal macrophages.

[The effect of glutoxim on Na+ transport in frog skin: the role of cytoskeleton].

Using voltage-clamp technique, the possible role of the cytoskeleton in the effect of pharmacological analogue of oxidized glutathione (GSSG), drug glutoxim, on Na+ transport in the frog Rana temporaria skin was investigated. It was shown for the first time that preincibation of the skin with the microtubular disrupter, nocodazole, actin filament disrupter, cytochalasin D or protein phosphatase PP1/PP2A inhibitor, calyculin A, significantly decrease the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of microtubules and microfilaments in the regulatory effect of glutoxim on Na+ transport in frog skin and that reorganization of actin filaments or microtubules leads to inhibition of stimulatory effect of glutoxim on Na+ transport in frog skin epithelia.

[The involvement of tyrosine kinases and phosphatidylinositol kinases in the effect of oxidized glutathione and glutoxim on Na+ transport in frog skin].

Using voltage-clamp technique, the role of tyrosine kinases and phosphatidylinositol kinases in the effect of oxidized glutathione (GSSG) and its pharmacological analogue, drug glutoxim, on Na+ transport in the frog Rana temporaria skin was investigated. It was shown for the first time that preincubation of the skin with tyrosine kinase inhibitor genistein or with two structurally distinct phosphatidylinositol kinase inhibitors, wortmannin and LY294002, significantly decreased the stimulatory effect of GSSG or glutoxim on Na+ transport. The data suggest that GSSG and glutoxim might transactivate insulin receptor in the basolateral membrane of epithelial cells and trigger the signaling cascade, involving tyrosine kinases and phosphatidylinositol kinases, which lead to Na+ transport stimulation in frog skin.

[The effect of oxidized glutathione and its pharmacological analogue, glutoxim, on intracellular Ca2+ concentration in macrophages].

The effect of oxidized glutathione (GSSG) and its pharmacological analogue, glutoxim, on intracellular Ca2+ concentration in rat peritoneal macrophages was investigated using Fura-2AM microfluorimetry. It was shown that both GSSG and glutoxim increased intracellular Ca2+ concentration inducing Ca(2+)-mobilization from thapsigargin-sensitive Ca(2+)-stores and subsequent Ca2+ entry into macrophages from external medium. Dithiothreitol, which reduces S-S-bonds in proteins, completely prevented or reversed the increase in the intracellular Ca2+ concentration induced by GSSG or glutoxim. It suggests that the increase in the intracellular Ca2+ concentration induced by GSSG or glutoxim can be mediated by their interactions with functionally important SH-groups of proteins involved in Ca(2+)-signaling. Two structurally different tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate, prevented or completely reversed the increase in the intracellular Ca(2+)-concentration induced by GSSG or glutoxim. On the contrary, tyrosine phosphatase inhibitor, Na orthovanadate, enhanced the increase in the intracellular Ca2+ concentration evoked by oxidizing agents. The data suggest that tyrosine kinases and tyrosine phosphatases are involved in regulatory effects of GSSG and glutoxim on the intracellular Ca2+ concentration in macrophages.

[The effect of phosphatidylinositol kinase inhibitors on store-dependent Ca2(+)-transport in macrophages].

Using Fura-2AM microfluorimetry the role of phosphatidylinositol kinases in the regulation of Ca2+ signals induced by purinergic agonist ATP and endoplasmic Ca2(+)-ATPase inhibitor thapsigargin in rat peritoneal macrophages was investigated. It was shown that two structurally distinct phosphatidylinositol 3- and phosphatidylinositol-4-kinases inhibitors wortmannin and LY294002 showed a dose- dependent effect on store-dependent Ca2(+)-entry, induced by thapsigargin or ATP. The data suggest that phosphatidylinositol 3- and phosphatidylinositol-4-kinases play an important role in the activation of store-dependent Ca2(+)-entry in macrophages and that their effect might be mediated by their influence on actin cytoskeleton. The results are compatible with the "secretion-like coupling model" for store-dependent Ca2(+)-entry in macrophages based on a reversible trafficking and coupling of the Ca2+ store with the plasma membrane which suggests the involvement of microfilaments and phosphatidylinositol kinases.

[Effect of latrunculin B, jasplakinolide and brefeldin A on the store-dependent Ca2+ entry in macrophages].

Using Fura-2AM microfluorimetry, effect of actin filament modifiers and vesicular trafficking inhibitor on the store-dependent Ca2+ entry induced by purinergic agonists (ATP, UTP) and endoplasmic Ca2+-ATPase inhibitors (thapsigargin, cyclopiazonic acid) in rat peritoneal macrophages was investigated. It was shown that inhibition of actin polymerization by latrunculin B had a biphasic time-dependent effect on Ca2+ entry, showing an initial potentiation followed by inhibition of Ca2+ entry. Moreover, addition of latrunculin B after induction of store-dependent Ca2+ entry inhibited the Ca2+ influx. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-dependent Ca2+ entry but did not modify this process after its activation. Vesicular transport inhibitor brefeldin A, which inactivates arfproteins, inhibited activation of store-dependent Ca2+ entry but did not alter this mechanism once being initiated. These data are compatible with the sectretion-like coupling model for store-dependent Ca2+ entry in macrophages based on a reversible trafficking and coupling of Ca2+ store with the plasma membrane.

[Structural and functional organization of Na+ transport in epithelial systems. I. Epithelial Na+ channels].

The review summarizes recent data on the structural and functional organization and regulation mechanisms of Na+ transport in epithelial systems. The review is focused on the structure, function, regulation and pathology of epithelial Na+ channels, which are critical for Na+ homeostasis maintenance and blood pressure control.

[Phosphoinositide metabolism and the formation of the calcium signal in cells]. / Metabolizm fosfoinozitidov i formirovanie kal'tsievogo signala v kletkakh.

The recent experimental data on the role of phosphoinositides in cellular Ca2+ signalling are reviewed. Mechanisms of receptor-mediated Ca2+ mobilization from the intracellular Ca2+ stores and pathways of Ca2+ entry in the plasma membrane are discussed.

[The structural-functional organization of G-proteins and their bound receptors]. / Strukturno-funktsional'naia organizatsiia G-belkov i sviazannykh s nimi retseptorov.

Recent data on the structure, function and regulation of different types of GTF-binding proteins (G-proteins), playing the key role in transmembrane signalling, and of G-protein-coupled receptors are summarized. Possible mechanisms involved in the receptor-G-protein coupling and G-protein-membrane connection are discussed.

[Mechanisms of Ca2+ signalling in cells]. / Mekhanizmy Ca2+ signalizatsii v kletkakh.

The review summarizes recent data and current opinions of the Ca2+ signal formation in cells. Mechanisms of Ca2+ mobilization from the intracellular Ca2+ stores are discussed along with the pathways of Ca2+ entry from the external medium.

[Structural-functional organization of cellular signal transduction systems]. / Strukturno-funktsional'naia organizatsiia signal'nykh sistem v kletkakh

The review summarizes recent data and current opinions of the structural and functional organization of the known signalling systems and their functional elements. A possible role of adenylate cyclase, phosphoinositide, guanylate cyclase, tyrosine kinase systems and also of arachidonic acid, its oxygenated derivatives and of other fatty acids in intracellular signalling processes is discussed.

[Effect of ethidium bromide on the electrical activity of nerve cells]. / Deistvie étidium bromida na élektricheskuiu aktivnost' nervnoi kletki.

The interaction of ethidium bromide with stretch receptor neurons of the crayfish has been studied. The fluorescent staining of the neuron axon membrane was shown to be accompanied with the inactivation of its electrical action potential generation capacity. Taking into account the high specificity of ethidium bromide fluorescence rising under condition of its complexation with nucleic acids especially, the existence of RNA in composition of axon membrane electrogenic channels is suggested. The modification of RNA by ethidium bromide may lead to the inactivation of electrogenic channels in the axon membrane.

[The role of protein kinase C in Na+ transport regulation in the skin of adult frogs and tadpoles of Rana temporaria]. / Rol' proteinkinazy C v reguliatsii transépitelial'nogo transporta Na+ v kozhe vzroslykh osobei i golovastikov liagushki Rana temporaria.

Using the voltage-clamp technique, a possible role of protein kinase C in regulation of Na+ transport in the skin of the frog Rana temporaria was investigated. It was shown that protein kinase C activator phorbol ester 12-myristate 13-acetate (PMA), applied to the apical surface of the skin, stimulated transepithelial Na+ transport, measured as amiloride-sensitive short-circuit current, and also increased such electrical characteristics of frog skin as the open-circuit potential and transepithelial conductance. PMA exerts a similar stimulation effect on Na+ transport across the tadpole skin. Specific inhibitors of protein kinase C, chelerythryne or H-7, almost fully prevented the PMA-induced stimulation of Na+ transport. These data support a concept that the response to PMA was indeed mediated by PKC activation. The results are compatible with the important role played by protein kinase C in regulation of transepithelial Na+ transport in the skin of R. temporaria.

[Effect of inhibitors of mitochondrial oxidative metabolism on Ca-responses induced by purinergic agonists and thapsigargin in rat peritoneal macrophages]. / Vliianie ingibitorov okislitel'nogo metabolizma mitokhondrii na Ca-otvety, indutsirovannye purinergicheskimi agonistami i tapsigarginom v peritoneal'nykh makrofagakh krysy.

Effects of two metabolic inhibitors, oligomycin and carbonyl cyanide m-fluorophenylhydrazone (FCCP), on Ca2+ signals induced by purinergic agonists and thapsigargin in Fura-2-loaded rat peritoneal macrophages was investigated. 1 microgram/ml oligomycin or 1 microM FCCP were shown to inhibit 200 microM ATP or 200 microM UTP-evoked Ca2+ entry in macrophages. Independently of their chemical structure and site of inhibition, both metabolic poisons also inhibit the store-dependent or "capacitative" Ca2+ influx stimulated by emptying the intracellular Ca2+ stores with endoplasmic Ca(2+)-ATPase inhibitor thapsigargin (0.5 microM). These data are compatible with the important role the energy level of the cell plays in the control of Ca2+ entry in rat peritoneal macrophages.

[2 pools of mitochondria in a single mechanoreceptor neuron]. / O dvukh pulakh mitokhondrii v odinochnom mekhanoretseptornom neirone

The microspectrofluorimetric technique was applied to measure the intracellular ratio of oxidized flavoproteins and NADH in isolated neuron preparation from the stretch receptor organ of the crayfish Astacus leptodactilus. Two pools of mitochondria were detected differing from each other in their functional activity in the single cell at the same time. The mitochondria of the pool are localized near the nucleus, those of the other one being placed along the cell membrane. Physiological treatments (stretch, calcium deficient media) produce different changes in the activity of mitochondria belonging to the different pools. It is suggested that these pools of mitochondria furnish with energy different functional mechanisms within the same cell.

[Role of protein kinase C in the regulation of Ca-responses induced by purinergic agonists and thapsigargin in rat peritoneal macrophages]. / Rol' proteinkinazy C v reguliatsii Ca-otvetov, indutsirovannykh purinergicheskimi agonistami i tapsigarginom v peritoneal'nykh makrofagakh krysy.

The effect of protein kinase C activating phorbol ester, phorbol-12-myristate-13-acetate (PMA), on purinergic agonists- and thapsigargin-induced Ca2+ signals in Fura-2 loaded rat peritoneal macrophages was investigated. PMA (100 ng/ml) was shown to inhibit 200 muM ATP- or 200 microM UTP-evoked Ca2+ entry in macrophages. Protein kinase C activation by PMA also inhibits the store-dependent or "capacitative" Ca2+ influx stimulated by emptying the intracellular Ca2+ stores with endoplasmic Ca(2+)-ATPase inhibitor thapsigargin (0.5 microM). Inhibition of entry by PMA was fully prevented by protein kinase C inhibitor 50 microM H-7. These data are compatible with the important role played by protein kinase C in the control of Ca2+ entry in rat peritoneal macrophages.

[Differences in the photodestruction parameters of flavin fluorescence in normal and tumor cells at a decreased pH of the incubation medium]. / Razlichia v parametrakh fotodestruktsii flavinovoi fluorestsentsii normal'nykh i opukholevykh kletok pri snizhenii pH sredy inkubatsii.

Further investigation of the peculiarities of flavin fluorescence photodestruction in malignant cells was made. In normal cells incubated in low pH (3.0-3.2) physiological solutions, the decrease in the oxidized flavoprotein fluorescence intensity under irradiation is the same as in normal pH condition, whereas in tumor cell in low pH solutions a significant increase in the photodestruction level was noticed. The cells isolated from foci of transformation, following treatment of 3T3 NIH fibroblasts with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, displayed the photodestruction parameters similar to those in tumor cells. A two-step analysis of cells is proposed for distinguishing between the normal and malignant cells.

[Characteristics of "photo depletion" of the green fluorescence of tumor cells]. / Ob osobennostiakh "fotovygoraniia" zelenoi fluorestsentsii nekotorykh opukholevykh kletok.

The green (oxidized flavoproteins) fluorescence intensity was found to increase during investigation of NADH and oxidized flavoproteins fluorescence with the use of optimal excitation of different fluorescence bands. This effect was observed under excitation with blue light (436 nm). It is suggested that in some malignant cells, the structure of flavoproteins (probably of mitochondrial ones) may be altered in the way of increasing the quantum yield under the action of light irradiation.

[Effect of arachidonic and other fatty acids on the intracellular calcium concentration and calcium signaling in peritoneal macrophages]. / Vliianie arakhidonovoi i drugikh zhirnykh kislot na vnutrikletochnuiu kontsntratsiiu Ca2+ i formirovanie Ca2+-signalov v peritoneal'nykh makrofagakh.

Effects of arachidonic and other fatty acids on the intracellular Ca2+ concentration ([Ca2+]i) in rat peritoneal macrophages was investigated. It has been shown that cis-polyunsaturated arachidonic and linoleic induce a significant and dose-dependent increase in [Ca2+]i, which is due to depletion of thapsigargin-sensitive Ca2+ store and to stimulation of Ca2+ entry from the extracellular medium. Pharmacological characteristics of Ca2+ entry induced by arachidonic acid appeared to be similar to those of store-dependent Ca2+ entry activated by thapsigargin or cyclopiazonic acid; Ca2+ entry is attenuated by the same Ca2+ channel inhibitors, by tyrosine kinase inhibitor genistein and epoxygenase inhibitor proadifen. Cis-monounsaturated oleic and saturated myristic acids appeared to be less effective and induced only a slight increase in [Ca2+]i at much higher concentrations. Arachidonic and other fatty acids can also stimulate Ca(2+)-ATPase in the macrophage plasma membrane. The data are compatible with the important role played by arachidonic and other free fatty acids in the regulation of [Ca2+]i in peritoneal macrophages.

[Phenylarsine oxide, a tyrosine phosphatase inhibitor, induces an increase in the intracellular concentration of calcium in rat peritoneal macrophages and human fibroblasts]. / Ingibitor tirozinfosfataz fenilarzinoksid indutsiruet uvelichenie vnutrikletochnoi kontsentratsii Ca2+ v peritoneal'nykh makrofagakh krysy i fibroblastakh cheloveka.

Using Fura-2 microfluorimetry, phenylarsine oxide (PAO) (10-50 microM), a potent tyrosine phosphatase inhibitor, was shown to induce a dose-dependent increase in the free Ca2+ intracellular concentration in rat peritoneal macrophages and human foreskin fibroblasts. The PAO-induced increase in [Ca2+]i is not due presumably to depletion of intracellular Ca2+ stores but to mainly a stimulation of Ca2+ entry from the extracellular medium. This PAO-activated Ca2+ entry is attenuated by the following pharmacological agents. Organic and inorganic Ca2+ channel blockers: (nifedipine, verapamil and Ni2+); nonselective cation channel blocker niflumic acid; tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate; SH-reagents dithiothreitol parachloromercuribenzoate and N-ethylmaleimide; arachidonic acid metabolism inhibitors 4-bromophenacyl bromide, indomethacin and caffeic acid; microtubule disrupters vinblastine, colchicine and colcemide. On the contrary, microfilament disrupters, cytochalasin B and phalloidin, enhance PAO-activated Ca2+ entry. Our data suggest that the dynamic balance between tyrosine kinase and phosphatase activity may play a central role in the maintenance of homeostatic levels of [Ca2+]i both in unstimulated cells and after agonist application.