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Store-dependent Ca2+ entry is a ubiquitous mechanism of regulated Ca2+ entry into eukaryotic cells. It is activated upon depletion of intracellular Ca2+ stores and is involved in the regulation of a wide range of cellular processes. To elucidate the pharmacological characteristics of store-dependent Ca2+ entry into cells, we studied the effect of YM-58483, pyrazole derivative immunosuppressant, on the store-dependent Ca2+ entry in rat peritoneal macrophages induced by the endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, as well as the disulfide-containing immunomodulators glutoxim and molixan. Using Fura-2AM, fluorescent calcium indicator, it has been shown for the first time that, in rat peritoneal macrophages, as well as in other cell types, pyrazole derivative YM-58483 effectively inhibits store-dependent Ca2+ entry and is a useful pharmacological tool to study the store-dependent Ca2+ entry in macrophages. The data obtained additionally confirm that Ca2+ entry induced by glutoxim or molixan is realized via the store-dependent mechanism.

Using Fura-2AM, a fluorescent Ca2+ indicator, we have shown for the first time that sigma-1 receptor antagonist - neuroleptic chlorpromazine - significantly inhibits the store-dependent Ca2+ entry induced by immunomodulators glutoxim as well as molixan and endoplasmic reticulum Са2+-ATPase inhi-bitors thapsigargin and cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Using the voltage-clamp technique, it has been shown for the first time that the neuroleptic drugs chlorpromazine and trifluoperazine, which act as sigma-1 receptor antagonists, attenuate Na+ transport in the frog skin epithelium. The results suggest that sigma-1 receptors may be involved in the regulation of transepithelial Na+ transport in frog skin.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor antagonist neuroleptic chlorpromazine significantly inhibits glutoxim- and molixan-induced Ca2+ responses and Ca2+ responses induced by endoplasmic reticulum Са2+-ATPase inhibitors thapsigargin and cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in the signaling cascade induced by glutoxim or molixan and leading to intracellular Ca2+ concentration increase and in the regulation of store-dependent Ca2+ entry in macrophages.

Sigma-1 receptors are ubiquitous multifunctional ligand-regulated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Sigma-1 receptors bind ligands of different chemical structure and pharmacological action and modulate a wide range of cellular processes in health and disease, including Ca

Using Fura-2AM microfluorimetry we have shown for the first time that sigma-1 receptor agonist - tricyclic antidepressant amitriptyline - significantly inhibits store-dependent Ca2+ entry, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, in rat peritoneal macrophages. The results suggest possible involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor agonist-tricyclic antidepressant amitriptyline-significantly inhibits store-dependent Ca2+ entry, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, in rat peritoneal macrophages. The results suggest a possible involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with sigma-1 receptor antagonist haloperidol leads to a significant inhibition of the store-dependent Ca2+ entry induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of the sigma-1 receptor in the regulation of storedependent Ca2+ entry in macrophages.

Using voltage-clamp technique, the involvement of sigma-1 receptors in the regulation of Na+ transport in frog skin by the immunomodulatory drug glutoxim was investigated. We have shown for the first time that preincubation of the frog skin with the sigma-1 receptor antagonists haloperidol and chlorpromazine attenuates the stimulatory effect of glutoxim on the Na+ transport. The results suggest the possible involvement of the sigma-1 receptors in the regulation of Na+ transport in frog skin epithelium by glutoxim.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor agonist, tricyclic antidepressant amitriptyline, significantly inhibits glutoxim- and molixan-induced Ca2+-responses in rat peritoneal macrophages. The results suggest possible involvement of sigma-1 receptors in the signaling cascade induced by glutoxim or molixan and leading to intracellular Ca2+ concentration increase in macrophages.

Using voltage-clamp technique, the involvement of epoxygenases in immunomodulatory drug glutoxim regulation of Na+ transport in frog skin was investigated. We have shown for the first time that preincubation of the frog skin with epoxygenase inhibitors econazole or proadifen almost completely inhibits the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of the enzymes and/or products of epoxygenase oxidation pathway of arachidonic acid metabolism in glutoxim effect on Na+ transport in frog skin epithelium.

Using Fura-2AM microfluorimetry, it was shown for the first time that phospholipase A2 inhibitors 4-bromophenacyl bromide and glucocorticosteroids prednisolone and dexamethasone attenuate Ca2+ responses induced by neuroleptic trifluoperazine in macrophages. The results suggest the involvement of phospholipase A2 and arachidonic acid metabolism cascade in the effect of trifluoperazine on intracellular Ca2+ concentration in macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with the calsequestrin inhibitor neuroleptic trifluoperazine leads to a significant inhibition of the store-dependent Ca2+ entry induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest calsequestrin involvement in the regulation of the store-dependent Ca2+ entry in macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor antagonist neuroleptic chlorpromazine significantly inhibits glutoxim- and molixan-induced Ca

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor agonist-tricyclic antidepressant amitriptyline-significantly inhibits store-dependent Ca

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.

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.

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, leads to significant inhibition of thapsigargin-induced store-dependent Ca2+ entry in rat peritoneal macrophages. In contrast, macrophage treatment with methyl-β-cyclodextrin after Ca2+ entry mechanisms were activated by store depletion by thapsigargin application leads to potentiation of subsequent store-dependent Ca2+ entry. The results suggest that intact lipid rafts are necessary for the activation but not the maintenance of store-dependent Ca2+ entry in macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor antagonist, antipsychotic haloperidol, significantly inhibits glutoxim- and molixan-induced Ca2+-response in peritoneal macrophages. These results indicate possible involvement of sigma-1 receptors in the signal cascade induced by glutoxim or molixan and leading to intracellular Ca2+ concentration increase in macrophages.

Using voltage-clamp technique, the involvement of sigma-1 receptors in the regulation of Na

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor agonist, tricyclic antidepressant amitriptyline, significantly inhibits glutoxim- and molixan-induced Ca

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with sigma-1 receptor antagonist haloperidol leads to a significant inhibition of the store-dependent Ca

Using voltage-clamp technique, the involvement of epoxygenases in immunomodulatory drug glutoxim regulation of Na

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.

Using Fura-2AM microfluorimetry, we have shown for the first time that 5-lipoxygenase specific inhibitor antiasthmatic agent zileuton significantly inhibits Ca(2+)-responses induced by glutoxim and molixan in macrophages. The results support 5-lipoxygenase involvement in the effect of glutoxim and molixan on intracellular Ca(2+) concentration in macrophages and indicate the inadvisability of a combined use of drugs glutoxim and molixan and antiasthmatic agent zileuton.

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with the calsequestrin inhibitor neuroleptic trifluoperazine leads to a significant inhibition of the store-dependent Ca

Using Fura-2AM microfluorimetry, it was shown for the first time that phospholipase A

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, leads to significant inhibition of thapsigargin-induced store-dependent Ca

Using Fura-2AM microfluorimetry, it was shown for the first time that neuroleptic chlorpromazine causes intracellular Ca

Using voltage-clamp technique, the involvement of lipoxygenases in the effect of immunomodulatory drug glutoxim on Na

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor antagonist, antipsychotic haloperidol, significantly inhibits glutoxim- and molixan-induced Ca

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 Ca

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.

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 Rana temporaria 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.

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 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.

Using Fura-2AM microfluorimetry the possible involvement of epoxygenase pathway of arachidonic acid metabolism in the effect of glutoxim and molixan on intracellular Ca2+ concentration in rat peritoneal macrophages was investigated. It was shown for the first time that preincubation of the macrophages with epoxygenase inhibitors, proadifen and econazole, significantly decreases the intracellular Ca2+ concentration increase induced by glutoxim and molixan. The addition of the epoxygenase inhibitors during the already developed store-dependent Ca(2+)-entry induced by glutoxim or molixan partially inhibits Ca(2+)-entry. The obtained data suggest the involvement of the products and/or enzymes of epoxygenase pathway of the arachidonic acid metabolism in the glutoxim and molixan effect on the Ca2+ signaling processes in macrophages.

Using voltage-clamp technique, the involvement of WASP proteins and Arp2/3 complex in the effect of immunomodulator drug glutoxim on Na(+) transport in frog skin was investigated. It was shown for the first time that preincubation of the skin with the N-WASP inhibitor wiskostatin or the Arp2/3 complex inhibitor CK-0944666 significantly decreases the stimulatory effect of glutoxim on Na(+) transport. The data suggest the involvement of actin filament polymerization and branching in the glutoxim effect on Na(+) transport in frog skin.

Using the fluorescent Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

The Fura-2AM fluorescent Ca(2+) probe was used to study the possibility that the Arp2/3 complex and WASP proteins are involved in the effects of glutoxim and molixan on the intracellular Ca(2+) concentration in macrophages. It has been demonstrated that preincubation of macrophages with inhibitors of the Arp2/3 complex or WASP proteins (CK-0944666 or wiskostatin, respectively) results in a significant suppression of Ca(2+)-responses induced by glutoxim or molixan. This suggests that polymerization of actin filaments is a process involved in the effect of glutoxim or molixan on intracellular Ca(2+) concentration in macrophages.

Currently, a great number of disulfidecontaining drugs altering the redox status and having a physiolog� ically significant effect on cells have been developed and introduced into clinical practice. The pharmaceu� tical agent Glutoxim®, disodium salt of oxidized glu� tathione (GSSG) with a platinum nanoadditive (PHARMAVAM, Moscow, Russia) is used as an immunomodulator and a hemostimulant in the inte� grated therapy of bacterial and viral diseases, psoriasis, as well as radioand chemotherapies of cancer (1). Another disulfidecontaining agent, molixan (a com� plex of glutoxim and inosine nucleoside), has a similar application. However, the mechanisms of the cellular and molecular effects of these drugs are not com� pletely understood. We were the first to find out earlier that GSSG, glu� toxim, and molixan enhance the intracellular Са 2+