Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils.

Abstract

Stimulation of rabbit neutrophils by the chemotactic factors fMet-Leu-Phe and leukotriene B4, by platelet activating factor, or by arachidonic acid produces a rapid and dosedependent increase in the amounts of actin and of a 65,000-mol-wt protein associated with the cytoskeleton. Phorbol 12-myristate, 13-acetate, the calcium ionophore A23187 in the presence or absence of EGTA, and the fluorescent calcium chelator quin-2 also cause an increase in cytoskeletal actin. The stimulated increases in the cytoskeletal actin are not dependent on a rise in the intracellular concentration of free calcium and are not mediated by an increase in the intracellular pH or activation of protein kinase C. The increases in the cytoskeletal actin produced by fMet-Leu-Phe and leukotriene B4, but not by phorbol 12-myristate, 13-acetate, are inhibited by high osmolarity. The effect of hyperosmolarity requires a decrease in cell volume, is not mediated by an increase in basal intracellular concentration of free calcium, and is not prevented by pretreating the cells with amiloride. Preincubation of the cells with hyperosmotic solution also inhibits degranulation produced by all the stimuli tested. The inhibitory action of high osmolarity on the fMet-Leu-Phe and leukotriene B4 induced stimulation of cytoskeletal actin is discussed in terms of the possibility that the addition of high osmolarity, either directly or through activation of protein kinase C, causes receptor uncoupling.Most of the neutrophil responses that are induced by chemotactic factors, such as cell motility, shape change, projection of pseudopodia or ruffles, phagocytosis, and aggregation depend on the mechanical displacement of part or all of the cell. Accordingly, the cellular contractile apparatus, of which actin and myosin are the major components, must be closely involved in these responses. An understanding of neutrophil activation thus requires a detailed knowledge of the organization of these proteins before and after stimulation. Actin filaments in neutrophils, as in most nonmuscle cells, are considerably more labile than their counterpart in muscle, and large pools of depolymerized actin are usually found in resting cells (10,16,22).It has been found that the chemotactic responses of neutrophils are inhibited when the cells are incubated in hyperosmotic buffered solutions (2, 9). The inhibition of the functions of neutrophils by high osmolarity is particularly pertinent to areas of the kidney and urinary tract, where solute concentrations such as NaC1, urea, and glucose (in the case of diabetics) are high. It has been suggested that the susceptibility to infection of the medulla region of the renal system is mediated by the inhibitory effect on neutrophil functions of the hyperosmotic medullary environment (1,7,9). The molecular basis of this inhibition is not known at the present.Recently, it has been shown that the addition of the chemotactic factor fMet-Leu-Phe to neutrophils causes actin polymerization (5,17,26,28). This increase in polymerized actin...

Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils.

“…We sought to obtain more direct evidence for such a relationship by measuring respiratory burst oxidase activity in cytoskeleton preparations obtained -from PMA-activated human neutrophils. For our studies, cytoskeleton was prepared by extracting the cells with nonionic detergents using techniques similar to those employed in other laboratories (10)(11)(12). We found that the treatment of whole activated neutrophils with Triton X-100 brought most of the proteins into solution, but left behind an insoluble residue of detergent-resistant cytoskeleton.…”

“…4 ml of this suspension was incubated at 370C for 5 min, then supplemented with 40 ,d PMA solution (20 ,sg/ml in DMSO) and incubated for an additional 2 min at 370C to activate the neutrophils, terminating the reaction by immersion in an ice slurry. The suspension was centrifuged at 100 g for 10 min at 40C, the supernatant discarded, and the cells (total 4 X 107 neutrophils) suspended in 2 ml of Triton inhibitor buffer (10)(11)(12) and incubated at 0°C for 30 min. Next, 0.3 ml of the cell suspension was reserved for measuring total respiratory burst oxidase activity, while 0.…”

Phosphorylation and proteolytic modification of specific cytoskeletal proteins in human neutrophils stimulated by phorbol 12-myristate 13-acetate.

“…The cytoskeletal fraction was isolated by a modification (26) of the procedure reported by Yassin et al (27), which is basically an isotonic extraction in 2% Triton X-100 containing EGTA. For phosphorylation the cytoskeletal fraction containing 0.1 mg of protein was incubated with continuous shaking at 30QC in 0.25 ml of 50 mM sodium borate buffer, pH 7.5, containing 5 mM MgCl2 and 5 ,uM [y-32P]ATP (0.25 mCi/mmol) in the presence of 1.25 units of either protein kinase C or PKC-M.…”

“…Human neutrophils (5 x 106 cells per ml) prepared as previously described (22) were incubated at 37°C in 10 mM Hepes, pH 7.4, containing 0.14 M NaCl, 5 mM KCl, and 5 mM glucose, with or without PMA at 10-100 ng/ml, for 10 min. The cells were then cooled at 0°C and collected by centrifugation at 600 x g for 5 min.The cytoskeletal fraction was isolated by a modification (26) of the procedure reported by Yassin et al (27), which is basically an isotonic extraction in 2% Triton X-100 containing EGTA. For phosphorylation the cytoskeletal fraction containing 0.1 mg of protein was incubated with continuous shaking at 30QC in 0.25 ml of 50 mM sodium borate buffer, pH 7.5, containing 5 mM MgCl2 and 5 ,uM [y-32P]ATP (0.25 mCi/mmol) in the presence of 1.25 units of either protein kinase C or PKC-M.…”

Changes in neutrophil right‐angle light scatter can occur independently of alterations in cytoskeletal actin

“…The stimulation of PMNs with f-MLP results in a well-characterized biphasic RALS (6,13,16,39,40) and actin (7,8,16,33,(36)(37)(38) response. However, in comparing the data obtained by the two methods, similarities and differences are apparent.…”