V. G. Antonov

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.

Inhibitors of the cyclooxygenase oxidation pathway of arachidonic acid suppress the stimulating effect of glutoxim on Na(+) transport in frog skin.

193 The amphibian skin and other isolated epithelial systems are classical model objects for investigation of transepithelial ion transport. The amphibian skin and urinary bladder are similar to distal portions of the renal tubules in their transport of electrolyte and reacc tions to some hormones [1], which allows using data obtained on these objects for clarification of mechaa nisms of water and ion transport in renal cells. Recently, new disulfideecontaining agents with dd metals as nanoadditives, altering the cell redox state, have been widely used in clinical practice. Thus, the drug Glutoxim® (disodium salt of oxidized gluthathione (GSSG) with a platinum nanoadditive; PHARMAAVAM, Moscow, Russia) has been widely applied in clinical practice as an immunomodulator and a hemostimulant in the integrated therapy of bacc terial and viral diseases and psoriasis, as well as radioo and chemotherapies of cancer [2]. We have demonstrated earlier that Na + transport in the frog skin is modulated by various oxidizing agents. It has been demonstrated that GSSG and glutoxim applied to the basolateral surface of the frog skin imii tate the effect of insulin and stimulate transepithelial Na + transport [3]. Henceforth, it has been shown that tyrosine kinases, phosphatidylinositol kinases [4, 5], protein kinase C [6], serine/threonine protein phoss phatases PP1/PP2A [7], as well as microtubules and actin filaments [7, 8], are involved in the GSSG and glutoxim regulation of Na + transport in the frog skin, as well as in signaling cascades triggered by insulin. However, the molecular mechanisms underlying the regulatory effect of GSSG and glutoxim on the Na + transport remain unclear. It is known that arachidonic acid (AA) and its derivatives are important signaling molecules acting as local hormones and transmitters, playing a key role in the regulation of various physiological and pathophyss iological processes [9]. At the same time, AA is one of the major intracellular messengers, mediating various effects of insulin [10]. AA and its derivatives (mainly the products of cyclooxygenase oxidation pathway of AA, prostaglandins) are involved in the regulation of the ion and water transport in kidneys and other reabb sorbing epithelia, particularly, the frog skin epithelium [11]. Prostaglandins stimulate the transepithelial Na + transport, enhance the secretion of К + ions, and increase the water permeability of the apical memm brane in the frog skin [11]. Moreover, it has been found that the cyclooxygenase inhibitors diclofenac and ibuu profen suppress the Na + transport …

Lipoxygenases modulate the effect of glutoxim on Na+ transport in the frog skin epithelium

Using voltage-clamp technique, the involvement of lipoxygenases in the effect of immunomodulatory drug glutoxim on Na+ transport in frog skin was investigated. It was shown for the first time that preincubation of the skin with lipoxygenase inhibitors caffeic acid, baicalein, and nordihydroguaiaretic acid significantly decreases the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of lipoxygenase oxidation pathway of arachidonic acid metabolism in the glutoxim effect on Na+ transport in frog skin epithelium.

The effect of chlorpromazine on intracellular Ca 2+ concentration in macrophages

Using Fura-2AM microfluorimetry, it was shown for the first time that neuroleptic chlorpromazine causes intracellular Ca2+ concentration increase in macrophages due to Ca2+ mobilization from intracellular Ca2+ stores and subsequent Ca2+ entry from the external medium. Chlorpromazine-induced Ca2+ entry is inhibited by La3+ and 2-aminoethoxydiphenyl borate and is associated with Ca2+ store depletion.

Methyl-β-cyclodextrin inhibits Ca(2+)-responses induced by glutoxim and molixan in macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, significantly inhibits glutoxim- and molixan-induced Ca2+-responses in rat peritoneal macrophages. The results suggest that intact rafts are necessary for signaling cascade induced by glutoxim or molixan and leading to intracellular Ca2+ concentration increase in macrophages.

Phospholipase A2 inhibitors modulate the effects of glutoxim and molixan on the intracellular Ca2+ level in macrophages

Using the fluorescent Ca2+ probe Fura-2AM, the possible involvement of phospholipase A2, the key enzyme in the arachidonic acid cascade, in the effect of drugs glutoxim and molixan on the intracellular Ca2+ concentration in macrophages was studied. It was shown for the first time that preincubation of macrophages with the classical phospholipase A2 inhibitor, 4-bromophenacyl bromide, as well as with glucocorticosteroids prednisolone and dexamethasone significantly inhibits Ca2+ responses induced by glutoxim or molixan in macrophages. These results indicate the involvement of phospholipase A2 and arachidonic acid cascade in glutoximand molixan-induced signaling in macrophages.

Involvement of small G proteins and vesicle traffic in the glutoxim and molixan effects on the intracellular Ca(2+) concentration in macrophages.

252 Glutoxim (bisodium salt of oxidized glutathione (GSSG) containing nanoconcentrations of cissplatii num; PharmaVAM, Russia) is a pharmacological GSSG analogue used as an immunomodulating and hematopoesissstimulating agent in the complex therr apy of bacterial and viral infections, psoriasis, and in radioo and chemotherapy of malignant tumors [1]. Molixan (PharmaVAM) is a complex of glutoxim and inosine with an antiviral, immunomodulating, and hepatoprotective action used in the therapy of acute viral hepatitis B and C, mixed hepatitis, and liver cirr rhosis [1]. Glutoxim and molixan belong to the pharr macological group of thiopoetines, which affect intraa cellular redox regulation. However, the cellular and molecular mechanisms of their action are insuffii ciently understood. In our previous studies, it was first shown that GSSG, glutoxim, and molixan increased the intracell lular Ca 2+ concentration ([Ca 2+ ] i) in rat peritoneal macrophages by mobilizing calcium ions from thapsii garginnsensitive Ca 2+ stores and subsequently stimuu lating the Ca 2+ uptake [2–4]. Using a wide range of agents affecting different components of intracellular signaling systems, we first identified the principal elements of the signal cascade triggered by GSSG and glutoxim and resulting in a [Ca 2+ ] i increase in macrophages, namely, tyrosine kinases and tyrosine phosphatases [3, 5], phosphatii dylinositol kinases [6], and the key enzymes of the phosphoinositide signaling system, phospholipase C and protein kinase C [7]. It was also found that the effects of glutoxim and molixan on [Ca 2+ ] i in macc rophages were mediated by actin cytoskeleton elee ments [8] and microtubules [9]. The involvement of microtubules and the actin cytoskeleton in the glutoxim and molixan action on [Ca 2+ ] i in macrophages invites the assumption that macrophage activation induced by these agents is mediated by vesicle traffic. It is known that intracellu lar trafficking of secretory vesicles depends on microo tubules, which regulate the transport efficiency and organize the vesicle traffic by acting like cellular highh ways. Agents causing microtubule disintegration have been shown to inhibit secretion in different types of cells [10]. In addition, it was reported that glutoxim could induce vesicle exocytosis in macrophages conn taining M. tuberculosis [11]. Based on these data, we considered it worthwhile to investigate the possible involvement of vesicle transport and small G proteins, important components of the exocytosis signaling pathway, in mediating the glutoxim and molixan effects on the [Ca 2+ ] i level in macrophages. Experiments were …

Microtubular disrupter nocodazole and vesicular transport inhibitor brefeldin A attenuate the glutoxim effect on Na+ transport in frog skin

Using the voltage-clamp technique, a possible role of microtubules and vesicular transport in the effect of pharmacological analogue of oxidized glutathione, drug glutoxim, on Na+ transport in the frog Ranatemporaria skin was investigated. It was shown for the first time that the disrupter of microtubules nocodazole or inhibitor of vesicular transport brefeldin A similarly modulate (completely inhibit) the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of reorganization of microtubules and vesicular transport in the regulatory effect of glutoxim on Na+ transport.

Cyclooxygenase and lipoxygenase inhibitors modulate the Glutoxim and Molixan effects on the intracellular Ca 2+ concentration in macrophages

277 Glutoxim (disodium salt of GSSG with cisplatin at nano concentration, FARMA VAM, Moscow), a pharmacological analogue of oxidized glutathione (GSSG), is used as an immunomodulator and hemo stimulator in integrated treatment of bacterial and viral diseases and psoriasis, as well as in radiation ther apy and chemotherapy of cancer [1]. Another disul fide containing drug, Molixan (a complex of Glu Cyclooxygenase and Lipoxygenase Inhibitors Modulate the Glutoxim and Molixan Effects on the Intracellular Ca2+ Concentration in Macrophages Z. I. Krutetskaya, L. S. Kurilova, V. G. Antonov, and Academician A. D. Nozdrachev Received: April 1, 2013

Involvement of microtubules in the glutoxim regulation of Na(+) transport in the frog skin.

227 The amphibian skin and other isolated epithelial systems are classical model objects for studying the mechanisms of transepithelial ion transport. In their ability to transport electrolytes and in response to some hormones, the amphibian skin and bladder are similar to the distal renal tubules [1]; this allows the data for these model objects to be used for clarifying the mechanisms of water and ion transport in renal cells. The Na + transport in osmoregulatory epithelia is a complex multicomponent system providing the establishment and maintenance of electrolytic and water homeostasis. The key sodium transport proteins, such as amilorideesensitive epithelial Na + channels are targets for oxidative stress [2]. However, the mechh anisms underlying the effect of oxidants and reducing agents on individual components of the Na + transepii thelial transport are still unknown. Recently, new disulfideecontaining agents with ddmetals as nanoadditives, altering cell redox state, have been widely used. In particular, the drug Gluu toxim ® (disodium salt of oxidized gluthathione, GSSG, with platinum nanoaddition; FARMAVAM, Moscow, Russia) has been introduced into clinical practice as an immunomodulator and a hemostimulant in the integrated therapy of bacterial and viral diseases, psoriasis, as well as radioo and chemotherapies of cann cer [3]. We earlier demonstrated that the Na + transport in the frog skin was modulated by various oxidants. This was the first finding to demonstrate that GSSG and glutoxim applied to the basolateral frog skin surface imitate the effect of insulin and stimulate Na + transepp ithelial transport [4]. Later, it was shown that tyrosine kinases, phosphatidylinositol kinases [5, 6], protein kinase C [7], serine/threonine protein phosphatases PP1/PP2, and actin filaments [8] are involved in the glutoxim regulation of Na + transport in the frog skin. It is known that microtubules, similar to actin microfilaments, have a high redox sensitivity and are easily glutathionylated [9]. In addition, destruction of microtubules inhibits a stimulatory effect of several hormones on the Na + transport and prevents developp ment of adaptive responses in osmoregulatory epithee lial cells [10]. Correspondingly, it was reasonable to study the possible role of microtubules in the glutoxim regulation of Na + transport in the frog skin. Two strucc turally different microtubule depolymerizing agents, nocodazole and colcemid, were used in the experii ments, as well as taxol, a microtubuleestabilizing agent. The experiments were performed with male frogs Rana temporaria from October to March. The abdomm inal skin was cut …