The involvement of actin cytoskeleton in glutoxim and molixan effect on intracellular Ca<Superscript>2+</Superscript>-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.     

Methyl-β-cyclodextrin inhibits Ca<Superscript>2+</Superscript>-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.     

Amitriptyline Attenuates Ca<Superscript>2+</Superscript> Responses Induced by Glutoxim and Molixan in Macrophages

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.     

5-Lipoxygenase inhibitor zileuton inhibits Ca<Superscript>2+</Superscript>-responses induced by glutoxim and molixan in macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that 5-lipoxygenase specific inhibitor antiasthmatic agent zileuton significantly inhibits Ca²⁺-responses induced by glutoxim and molixan in macrophages. The results support 5-lipoxygenase involvement in the effect of glutoxim and molixan on intracellular Ca²⁺ concentration in macrophages and indicate the inadvisability of a combined use of drugs glutoxim and molixan and antiasthmatic agent zileuton.     

Sigma-1 receptor antagonist haloperidol attenuates Ca<Superscript>2+</Superscript> responses induced by glutoxim and molixan 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.     

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

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.     

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

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

The effect of glutoxim on Na<Superscript>+</Superscript> 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.     

The effect of oxidized glutathione and its pharmacological analogue glutoxim on intracellular Ca<Superscript>2+</Superscript> concentration in macrophages Ca<Superscript>2+</Superscript>

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 role of cytoskeleton structures in regulation of Ca2+ responses in macrophages

The role of the cytoskeleton in regulation of purinergic agonist- and endoplasmic Ca(2+)-ATPase inhibitors-induced Ca2+ signals in rat peritoneal macrophages was investigated. It has been shown that in cells pretreated with agents that disrupt microtubules (vinblastine, colchicine, colcemid) or actin microfilaments (cytochalasins, phalloidin), the ability of thapsigargin or cyclopiazonic acid to empty Ca2+ stores and activate store-dependent Ca2+ influx was significantly attenuated. On the contrary, microfilaments and microtubule disrupters did not affect ATP- or UTP-induced Ca2+ mobilization, indicating that release of Ca2+ from intracellular stores through the inositol phosphate pathway was intact. The results suggested that an intact cytoskeleton is required for capacitative Ca2+ entry but not for agonist-induced Ca2+ mobilization.     

The effect of phosphatidylinositol kinase inhibitors on store-dependent Ca2(+)-transport in macrophages

Using Fura-2AM microfluorimetry the role of phosphatidylinositol kinases in the regulation of Ca2+ signals induced by purinergic agonist ATP and endoplasmic Ca2(+)-ATPase inhibitor thapsigargin in rat peritoneal macrophages was investigated. It was shown that two structurally distinct phosphatidylinositol 3- and phosphatidylinositol-4-kinases inhibitors wortmannin and LY294002 showed a dose- dependent effect on store-dependent Ca2(+)-entry, induced by thapsigargin or ATP. The data suggest that phosphatidylinositol 3- and phosphatidylinositol-4-kinases play an important role in the activation of store-dependent Ca2(+)-entry in macrophages and that their effect might be mediated by their influence on actin cytoskeleton. The results are compatible with the "secretion-like coupling model" for store-dependent Ca2(+)-entry in macrophages based on a reversible trafficking and coupling of the Ca2+ store with the plasma membrane which suggests the involvement of microfilaments and phosphatidylinositol kinases.     

The organization of actin in spreading macrophages : The actin-cytoskeleton of peritoneal macrophages is linked to the substratum via transmembrane connections

The cytoskeleton of murine peritoneal macrophages has been examined by a combination of morphological techniques, including phase-contrast light microscopy, scanning electron microscopy (SEM), and several transmission electron microscopic (TEM) methods. The cytoskeleton of cells spreading on glass, Formvar-carbon, and polystyrene substrata was exposed by brief extraction with non-ionic detergent, and stabilized by exposure to heavy meromyosin, myosin subfragment-1 or tropomyosin. In the spreading lamellae and lamellipodia the cytoskeleton is principally composed of filamentous actin, which appears as dense foci, interconnected by radiating filaments and filament bundles. The actin of the foci, as well as individual actin filaments, are connected to the substratum by transmembrane linkages which appear as filaments that pass through the plane of the (extracted) plasma membrane. Thus, the results of this study indicate that the adhesion of macrophages to substrata for the purposes of spreading and motility may be a function of transmembrane elements which link actin to substrata. Further, the formation of actin foci may serve to stiffen and stabilize the cytoskeleton, conditioning it to function in cell adhesion, spreading and locomotion.     

Association of the actin cytoskeleton with glass-adherent proteins in mouse peritoneal macrophages

Summary— When mouse peritoneal macrophages adherent to glass surface were removed by treatment with triethanolamine and Nonidet P-40, fine thread structures of unique loops were left behind on glass at the sites of cell adhesion. To examine the ultrastructural relationship between such looped threads and cytoskeletal components in glass-adherent macrophages, we successfully used the ‘zinc method’ to remove most of the cytoplasm including nuclei and to expose the cytoskeleton associated with the ventral plasma membrane. The cytoskeleton was seen to be mainly composed of actin filaments forming dense networks. The network contained scattered star-like foci from which actin filaments radiated. When the ventral plasma membrane-cytoskeleton complex was further treated with Nonidet P-40, the membrane was dissolved to expose the glass surface with actin foci persisting on glass. When the complex was removed by further treatment with Nonidet P-40 and DNase I, the looped threads became visible. Confocal laser microscopy of glass-adherent macrophages stained with fluorescent phalloidin showed the preferential distribution of F-actin in the ventral cytoplasm along the plasma membrane, where intense fluorescent spots were also scattered. Confocal interference reflection microscopy revealed densely populated dark dots and striae of focal contact, which corresponded in overall distribution to actin foci and looped threads. These observations suggest that actin cytoskeleton is closely associated with looped threads to reinforce cell adhesion to glass.     

N-WASP has the ability to compensate for the loss of WASP in macrophage podosome formation and chemotaxis

Wiskott-Aldrich syndrome protein (WASP) and its homologue neural-WASP (N-WASP) are nucleation promoting factors that integrate receptor signaling with actin cytoskeleton rearrangement. While hematopoietic cells express both WASP and N-WASP, WASP deficiency results in altered cell morphology, loss of podosomes and defective chemotaxis. It was determined that cells from a mouse derived monocyte/macrophage cell line and primary cells of myeloid lineage expressed approximately 15-fold higher levels of WASP relative to N-WASP. To test whether N-WASP can compensate for the loss of WASP and restore actin cytoskeleton integrity, N-WASP was overexpressed in macrophages, in which endogenous WASP expression was reduced by short hairpin RNA (shWASP cells). Many of the defects associated with the loss of WASP, such as podosome-dependent matrix degradation and chemotaxis were corrected when N-WASP was expressed at equimolar level to that of the wild-type WASP. Furthermore, the ability of N-WASP to partially compensate for the loss of WASP may be physiologically relevant since activated murine WASP-deficient peritoneal macrophages, which show enhanced N-WASP expression, also show an increase in matrix degradation. Our study suggests that expression levels of WASP and N-WASP may influence their roles in actin cytoskeleton rearrangement and shed light to the complex intertwining roles WASP and N-WASP play in macrophages.     

Role of actin cytoskeleton in LPS-induced NF-kappaB activation and nitric oxide production in murine macrophages

Lipopolysaccharide (LPS) is a major cell wall component of Gram-negative bacteria and is known to cause actin cytoskeleton reorganization in a variety of cells including macrophages. Actin cytoskeleton dynamics influence many cell signaling pathways including the NF-kappaB pathway. LPS is also known to induce the expression of many pro-inflammatory genes via the NF-kappaB pathway. Here, we have investigated the role of actin cytoskeleton in LPS-induced NF-kappaB activation and signaling leading to the expression of iNOS and nitric oxide production. Using murine macrophages, we show that disruption of actin cytoskeleton by either cytochalasin D (CytD) or latrunculin B (LanB) does not affect LPS-induced NF-kappaB activation and the expression of iNOS, a NF-kappaB target gene. However, disruption of actin cytoskeleton caused significant reduction in LPS-induced nitric oxide production indicating a role of actin cytoskeleton in the post-translational regulation of iNOS.     

Effects of some biologically active compounds on phagosome-lysosome fusion in peritoneal macrophages of mice.

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P-L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P-L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes. All three compounds tested increase F-actin content in peritoneal macrophages. Thus, the effect of BR on P-L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P-L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.     

Magnetic Field Changes Macrophage Phenotype

Macrophages play a crucial role in homeostasis, regeneration, and innate and adaptive immune responses. Functionally different macrophages have different shapes and molecular phenotypes that depend on the actin cytoskeleton, which is regulated by the small GTPase RhoA. The naive M0 macrophages are slightly elongated, proinflammatory M1 are round, and M2 antiinflammatory macrophages are elongated. We have recently shown in the rodent model system that genetic or pharmacologic interference with the RhoA pathway deregulates the macrophage actin cytoskeleton, causes extreme macrophage elongation, and prevents macrophage migration. Here, we report that an exposure of macrophages to a nonuniform magnetic field causes extreme elongation of macrophages and has a profound effect on their molecular components and organelles. Using immunostaining and Western blotting, we observed that magnetic force rearranges the macrophage actin cytoskeleton, the Golgi complex, and the cation channel receptor TRPM2, and modifies the expression of macrophage molecular markers. We have found that the magnetic-field-induced alterations are very similar to changes caused by RhoA interference. We also analyzed magnetic-field-induced forces acting on macrophages and found that the location and alignment of magnetic-field-elongated macrophages correlate very well with the simulated distribution and orientation of such magnetic force lines.     

Effect of alkaloid sanguinarine and a pharmaceutical preparation ukrain on modulation of vesicular membrane fusion and actin cytoskeleton of macrophages

A study was made of modulations of lysosome-phagosome fusion process and of fibrillar actin content in mouse peritoneal macrophages by an antitumor alkaloid sanguinarine and a derivative drug Ukrain. In addition, effects of these substances on in vitro polymerization of monomeric globular actin from rabbit muscle were investigated. Sanguinarine and Ukrain stimulated lysosome-phagosome fusion and increased the content of polymerized fibrillar form of actin in mouse macrophages. Effects of these substances were enhanced at their higher concentrations. Both sanguinarine and Ukrain induced in vitro polymerization of globular actin from rabbit muscle. A possible role of sanguinarine and Ukrain in changing vesicular membrane states during intracellular membrane interaction in lysosome-phagosome fusion process was discussed. The influence of these substances on actin polymerization and actin cytoskeleton rearrangement was evaluated. It could be supposed that sanguinarine and Ukrain may alter intracellular membrane transport.     

The inhibitors of Arp2/3 complex and WASP proteins modulate the effect of glutoxim on Na<Superscript>+</Superscript> transport in frog skin

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.     

双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞细胞骨架的影响

目的探讨双歧双歧杆菌的完整肽聚糖（WPG）对巨噬细胞细胞骨架的影响。方法首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞后,用荧光标记的鬼笔环肽染液染色,最后采用激光共聚焦显微镜技术检测巨噬细胞的细胞骨架。结果和对照组相比,WPG刺激巨噬细胞后,其胞内肌动蛋白减少且排列更加紊乱,同时胞膜荧光强度减弱,胞外放射状荧光物质减少,甚至消失。结论双歧双歧杆菌的WPG在激活巨噬细胞的过程中可影响其细胞骨架。