Involvement of microtubules in the effects of glutoxim and molixan on the intracellular concentration of Ca2+ in macrophages

Abstract

196Currently, a great number of disulfide containing 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 (PHARMA VAM, Moscow, Russia) is used as an immunomodulator and a hemostimulant in the inte grated therapy of bacterial and viral diseases, psoriasis, as well as radio and chemotherapies of cancer [1]. Another disulfide containing 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+ concentration ([Ca 2+ ] i ) due to Са 2+ mobilization from the thapsigargin sensitive Са 2+ stores and subsequent entry of Са 2+ into rat peritoneal macrophages [2][3][4]. Using a wide range of agents affecting the components of signaling systems in cells, we have demonstrated that tyrosine kinases, tyrosine phosphatases [3, 5], phosphatidylinositol kinases [6], small G proteins of the Ras superfamily, and the most important enzymes of the phosphoinositide system of signal transduction (phospholipase C and protein kinase C) [7] are the key players in the signaling cascade triggered by GSSG and glutoxim and leading to an increase in [Ca 2+ ] i in mac rophages. It has also been found that elements of the actin cytoskeleton are involved in the effects of glutoxim and molixan on [Ca 2+ ] i in macrophages [8,9].It is known that the microtubule protein tubulin has a high redox sensitivity and can be easily S glu tathionylated [10]. Correspondingly, it was reasonable to study the possible role of microtubules in the regu latory effect of glutoxim or molixan on [Ca 2+ ] i in the rat peritoneal macrophages.The procedure of macrophage culturing and an automated system for [Ca 2+ ] i recording with the use of the fluorescent probe Fura 2AM were described ear lier in detail [11]. The experiments were performed at a room temperature of 20-22°C on the second or third day of cell culturing. The involvement of micro tubules in the effects of glutoxim and molixan on [Ca 2+ ] i in the rat peritoneal macrophages was studied using two structurally different agents capable of inducing depolymerization of microtubules, colcemid and nocodazole, as well as the microtubule stabilizer taxol [12].It has been demonstrated that incubation of mac rophages in the presence of 100 µg/mL molixan (Fig. 1a) or 100 µg/mL glutoxim (Fig. 2a) for 20 min in a medium without calcium causes a significant increase in [Ca 2+ ] i , which reflects mobilization of Са 2+ from intracellular Са 2+ stores. Addition of 2 mM Са 2+ to the external medium induces entry of Са 2+ into the cytosol, which is apparently mediated by depletion of the Са 2+ store (Figs. 1a, 2a).It has been found that nocodazole, colcemid, or taxol almost complet...

Arp2/3 complex is involved in the effect of glutoxim and molixan on intracellular Ca2+ concentration in macrophages

“…Thus in T lymphocytes with defective protein WAVE2 activating the Arp2/3 complex one observes suppression of store dependent Ca 2+ entry activated by thapsigargin [22]. The above results, along with our previous findings [2][3][4][5][6][7][8][9][10][11] testify about that in the action of glutoxim or molixan on [Ca 2+ ] i in macrophages the same signal proteins and their complexes as in the process of exocytosis are involved: tyrosine kinases, G proteins of small molec ular mass, mechanisms of vesicular transport; actin and tubulin cytoskeleton, and also Arp2/3 complex mediating rearrangements of the actin cytoskeleton. Reorganization of the actin cytoskeleton induced in macrophages upon action of glutoxim or molixan may also mediate activation of macrophages and alleviate processes of endo and exocytosis.…”

“…Using a wide range of agents affecting components of intracellular signaling systems in cells, we have shown for the first time that key players in a signal cas cade triggered by GSSG and glutoxim and leading to [Ca 2+ ] i increase in macrophages are tyrosine kinases and tyrosine phosphatases [3,5], phosphatidylinositol kinases [6], key enzymes of the phosphoinositide sig nal pathway -phospholipase C and protein kinase C [7]. It was also found that the effect of glutoxim and molixan on [Ca 2+ ] i in macrophages was mediated by actin cytoskeleton [8] and microtubules [9].…”

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

“…The above results, along with our previous findings [2][3][4][5][6][7][8][9], also indicate that the action of glutoxim and molixan on [Ca 2+ ] i levels in macrophages involves the same signal proteins and complexes that participate in exocytosis: tyrosine kinases and tyrosine phos phatases, phosphatidylinositol 3 and 4 kinases, pro tein kinase C, small G proteins of the Ras superfamily, vesicle trafficking, and actin and tubulin cytoskeleton [15]. In addition, we have shown that glutoxim and molixan themselves induce reorganization of the actin cytoskeleton [8], which mediates macrophage activa tion and facilitates endo and exocytosis.…”

“…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].…”