Involvement of small G proteins and vesicle traffic in the glutoxim and molixan effects on the intracellular Ca2+ concentration in macrophages

Abstract

252Glutoxim (bisodium salt of oxidized glutathione (GSSG) containing nanoconcentrations of cis plati num; PharmaVAM, Russia) is a pharmacological GSSG analogue used as an immunomodulating and hematopoesis stimulating agent in the complex ther apy of bacterial and viral infections, psoriasis, and in radio 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 cir rhosis [1]. Glutoxim and molixan belong to the phar macological group of thiopoetines, which affect intra cellular redox regulation. However, the cellular and molecular mechanisms of their action are insuffi ciently understood.In our previous studies, it was first shown that GSSG, glutoxim, and molixan increased the intracel lular Ca 2+ concentration ([Ca 2+ ] i ) in rat peritoneal macrophages by mobilizing calcium ions from thapsi gargin sensitive Ca 2+ stores and subsequently stimu lating the Ca 2+ uptake [2][3][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], phosphati 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 mac rophages were mediated by actin cytoskeleton ele 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 micro tubules, which regulate the transport efficiency and organize the vesicle traffic by acting like cellular high 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 con 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 performed on a culture of resi dential peritoneal macrophages of Wistar rats at room temperature (20-22°C) 24-48 h after the beginning of cell culture. The procedures of macrophage cultur ing and the automated device for [Ca 2+ ] i measure ments based on a Leica DM 4000B fluorescent micro scope (Leica Microsystems, Germany) were previ ously described in detail [8]. The levels of [Ca 2+ ] i were measured using a Fura 2AM fluo...