Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils.

Abstract

In human peripheral blood polymorphonuclear leukocytes and lymphocytes, GSH-oxidizing agents promote the movement of surface-bound concanavalin A (Con A) into caps and inhibit the assembly of microtubules (MT) that is normally induced by Con A binding. Con A capping and inhibition of MT assembly occur when GSH levels in cell suspensions are decreased by 30-70%, and return of GSH to control levels is accompanied by the appearance of cytoplasmic MT and by inhibition of the capping response with Con A. Oxidation of GSH markedly stimulates the hexose monophosphate shunt, and regeneration of GSH occurs rapidly.The data indicate that MT cannot be assembled or maintained in the face of decreased GSH levels. Thus, GSH homeostasis becomes critical during physiological events such as phagocytosis which simultaneously induce the assembly of MT and the production of agents like H202 that can oxidize GSH.Although a great deal of information is presently available about the conditions required for microtubule (MT) assembly in vitro, there is still no clear understanding of how assembly is regulated in vivo. Nath and Rebhun (21) have recently shown that oxidation of GSH in fertilized sea urchin eggs inhibits mitotic spindle formation and causes dissolution of preformed spindles. We proposed that intracellular GSH may also influence the organization and function of cytoplasmic MT. To test this hypothesis, we examined the effects of GSH oxidation on the assembly of MT in human peripheral blood polymorphonuclear leukocytes (PMN). Two relatively specific oxidizing agents were employed: diamide (diazene dicarboxylic and bis-N,N-dimethylamide) (15, 16) and BHP (tertiary butylhydroperoxide) (30).PMN were chosen for study first because MT assembly can be reproducibly induced by brief exposure of a variety of ligands including the plant lectin concanavalin A (Con A) (8,10,25), and second because the presence or absence of MT in Con A-treated PMN can be inferred from the distribution of surface-bound lectin. Thus, Con A shows a homogeneous surface distribution on cells that are competent to assemble MT but moves into a single aggregate-a cap-on the surface of cells treated with agents that bind with tubulin subunits and inhibit their polymerization (colchicine and the new carbamate anti-MT agent, R17934) (22-27).

Microtubule assembly and conanavalin A capping in lymphocytes: reappraisal using normal and abnormal human peripheral blood cells.

“…These dissimilar agents both bind tubulin dimers and prevent their polymerization (9). Similar results have been obtained (5,8,10) before with the same and lower concentrations of these agents. As described (5,8), the majority of PMN were also readily capped after exposure to 100uM diamide for 10 min.…”

“…We also noted that T lymphocytes and small monocytes, which are difficult to distinguish by nuclear shape alone, can be separated on the basis of the distribution of bound lectin: after 10 (5,8) in which randomly selected centrioles were photographed at X17,300 magnification and enlarged 2.7-fold in printing, and the number of microtubules was determined with a 9.5-cm square centered on the centriole. This corresponded to an original 4 Mm2 area of the cell.…”

“…Buffy coat leukocytes were obtained from freshly drawn, heparinized blood and suspended in modified Hanks' medium as described (5,6). The leukocyte suspensions contained approximately 70-80% PMN leukocytes and 20-30% lymphocytes and monocytes.…”

The effect of apomorphine on exocytosis and metabolic burst of polymorphonuclear leukocytes

“…The PMNL contains in its cytoplasm a very high concentration (4mM) of glutathione (Oliver et al, 1976) which might be expected to inactivate apomorphine should it penetrate into the cell. The vulnerable sulphydryl groups involved are thus probably located on proteins of the PMNL plasma membrane.…”

Importance and Regulation of Hepatic Glutathione

“…Conversely, maintaining the normal concentration of reduced glutathione supports the maintenance of the reduced state of protein thiols. Formation of glutathione-protein mixed disulfides has been shown to alter a variety of cell functions, including enzyme function, protein synthesis, cell integrity, microtubular function, transport processes, and release mechanisms [1,5]. Since oxidized glutathione, when present in 497…”