O. E. Lebedev

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

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

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

Using the voltage-clamp technique, the possible implication of cytoskeleton in the effect of glutoxim, a pharmacological analog of oxidized glutathione (GSSG), on Na+ transport in the skin of frog Rana temporaria was investigated. It was shown for the first time that skin preincubation with nocodazole, a microtubular disrupter; cytochalasin D, actin filament disrupter; or protein phosphatase PP1/PP2A inhibitor calyculin A significantly decreased the stimulatory effect of glutoxim on Na+ transport. The results 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 the stimulatory effect of glutoxim on Na+ transport in frog skin epithelia.

Involvement of tyrosine and phosphatidylinositol kinases in oxidized glutathione and glutoxim regulation of Na+ transport in frog skin

The role of tyrosine and phosphatidylinositol kinases in oxidized glutathione (GSSG) and its pharmacological analogue, glutoxim, regulation of Na+ transport in Rana temporaria frog skin was investigated by the voltage-clamp technique. It was shown for the first time that the 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 can transactivate insulin receptor in the basolateral membrane of epithelial cells and trigger the signaling cascade, which involves tyrosine and phosphatidylinositol kinases, which stimulates Na+ transport in frog skin.

[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 Ca2+-concentration increase induced by glutoxim or molixan in rat peritoneal macrophages. Morphological data obtained with the use of rhodamine-phalloidine demonstrated that glutoxim and molixan cause the actin filaments reorganization in rat peritoneal macrophages.

The involvement of protein kinase C in the effect of oxidized glutathione and glutoxim on Na+ transport in frog skin

The amphibian skin and other isolated epithelial systems serve as classic model objects to study transepithelial ion transport mechanisms. Na + transport in epithelial cells is known to be a complex multicomponent system containing various Na + transporting proteins, which may be targets for oxidative stress. Previously, we have demonstrated that Na + transport in frog skin can be modulated by various oxidizing agents. It was shown for the first time that oxidized glutathione (GSSG) and its pharmacological analogue glutoxim applied to the basolateral surface of the frog skin imitated the effect of insulin and increased transepithelial Na + transport. Furthermore, we elucidated for the first time the involvement of tyrosine kinases and phosphatidylinositol kinases in the stimulatory effect of GSSG and glutoxim on Na + transport in frog skin. It is known that insulin interacts with the receptor with intrinsic tyrosine kinase activity located in the basolateral membrane of epithelial cells. Previously, we have demonstrated the involvement of tyrosine kinases, tyrosine phosphatases, phosphatidylinositol kinases and protein kinase C in the effect of insulin on Na + transport in frog skin. Therefore, the purpose of the present research was to study the possible role of protein kinase C in the regulatory effect of GSSG and glutoxim on Na + transport in the frog Rana temporaria skin. Using the voltage-clamp technique we studied the influence of the specific protein kinase C inhibitor calphostin C on the effect of GSSG and glutoxim on Na + transport in frog skin. To measure I-V relations, transepithelial potential V T was changed periodically to a series of nonzero values. From skin I-V relations the electrical characteristics of frog skin were determined: the short-circuit current I SC (I SC = I T at V T =0, where I T is the transepithelial current), the open-circuit potential (V OC = V T at the total transepithelial current I T = 0), and transepithelial conductance (g T ). The transepithelial Na + transport was measured as amiloride-sensitive I SC . It was shown that 100 µ g/ml GSSG or glutoxim, applied at the basolateral side of the skin, caused a significant increase of Na + transport. In a series of ten experiments I SC increased by 40 ± 11% ( P < 0.05) and 20 ± 1% ( P < 0.01), and V OC increased by 48 ± 10% ( P < 0.05) and 20 ± 1% ( P < 0.01) for GSSG and glutoxim, respectively. The value of g T did not change. It appeared that the inhibitor of protein kinase C calphostin C (1 µ M or 500 nM) significantly reduced the stimulatory effect of GSSG and glutoxim on Na + transport in frog skin. Thus, addition of 100 µ g/ml glutoxim to the basal side of the skin preincubated with calphostin C produced significantly lower changes of electrical characteristics values: I SC increased by 12 ± 1% ( P < 0.01) and 17 ± 1% ( P < 0.01), and V OC increased by 13 ± 2% ( P < 0.01) and 16 ± 1% ( P < 0.01) for 1 µ M and 500 nM calphostin C, respectively. Similar results were obtained when 100 µ g/ml GSSG was added to the basal side of the skin preincubated with calphostin C. The specific inhibitor of epithelial Na + channels (ENaC) amiloride (20 µ M), applied to the apical solution at the end of each experiment, inhibited I SC , suggesting that the effect of GSSG and glutoxim on Na + transport is mostly caused by modulation of the ENaC activity. Thus, we demonstrated for the first time the involvement of protein kinase C in the stimulatory effect of GSSG and glutoxim on Na + transport in the frog Rana temporaria skin. The results obtained in this study, as well as our earlier data, suggest that GSSG and glutoxim may interact with cysteine-rich domains of insulin receptor in the basolateral membranes of epithelial cells, transactivate it and trigger a complex signaling cascade, including tyrosine kinases, phosphatidylinositol kinases and protein kinase C. This leads to ENaC activation and Na + transport stimulation in frog skin.