A phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids.

Abstract

When rabbit peritoneal neutrophils were treated with glucocorticoids, their chemotactic response to stimulation by the chemoattractant fMet-Leu-Phe was markedly reduced. Preincubation of cells with glucocorticoids also decreased phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) activity in situ as measured by the release of [l-14C- Glucocorticoids exhibit a variety of biological effects, such as enzyme induction and anti-inflammatory actions (1, 2). The steroids act as a consequence of their binding to cytoplasmic receptors, followed by the translocation of the ligand-receptor complex into the nucleus, which, in turn, affects the transcription of various RNA species (3). The anti-inflammatory action of glucocorticoids is expressed in their ability to inhibit both chemotaxis and release of lysosomal enzymes in phagocytic cells (4-6). Recently, it has been demonstrated that chemotactic peptides enhance the release of arachidonic acid, a product of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4), in rabbit neutrophils (7). Steroids possibly exert their anti-inflammatory action by preventing the release of arachidonic acid from phospholipids and its conversion to prostaglandins (8). These observations prompted a search for a steroid-induced inhibitor of phospholipase A2 in neutrophils. Here, we report the existence of a protein in rabbit neutrophils that inhibits phospholipase A2 and whose synthesis is induced by glucocorticoids.METHODS AND MATERIALS Assay of Phospholipase A2 Activities in Situ and in Vitro. Rabbit peritoneal neutrophils were obtained as described (7) and treated with various glucocorticoids in RPMI 1640 medium (GIBCO) for 16 hr at 37°C under a humid atmosphere of 95% 02/5% CO2. Phospholipase A2 activity was measured by the release of ["4C]arachidonic acid from the cellular lipids, mainly phospholipids, with a slight modification of the method described (7). Bovine serum albumin (1%) was included in Gey's balanced salt solution buffered with 10 mM Hepes, pH 7.4 (modified Gey's buffer). The cells (8-11 X 106 cells per ml) were preincubated in a total volume of 5 ml with 1.25 ,uCi of [1-'4C]arachidonic acid (55.5 mCi/mmol) at 370C for 1 hr (1 Ci = 3.7 X 1010 becquerels). The cells were washed twice with 5 ml of modified Gey's buffer and resuspended in 5 ml of the same buffer. For arachidonic acid release, the cells were stimulated with 10 nM fMet-Leu-Phe for 10 min at 370C (7). 16 hr. After three washings with 0.84% NaCl solution buffered with 10 mM sodium phosphate buffer, pH 7.4, the cells (8 X 106 cells) were lysed in 3 ml of distilled water. After centrifugation, at 27,000 X g for 50 min, the precipitates were solubilized with 0.5 ml of 2% Nonidet P40. Samples (0.5 ml) of 2533The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Neutrophils from patients undergoing hip surgery exhibit enhanced movement under spinal anaesthesia compared with general anaesthesia

“…28 This effect takes two hours to develop. [29][30][31][32] It is possible that a cortisol depressant effect may have influenced the findings of Stanley et al, as at that sample time a neutrophilia was present in their patients. After 90 rain of anaesthesia and surgery, our patients did not show any alterations in total white counts compared with preoperative levels in any group probably due to a shorter duration of anaesthesia prior to the commencement of surgery in all groups.…”

Inhibition of human cytosolic phospholipase A2 by human annexin V

“…The discovery of the lipocortins, identified by their being inhibitors of phospholipase A # (PLA # ) [1,2], provided some of the first structural and functional information that led to the identification of a large group of proteins now known as the annexins [3]. Both annexins and phospholipases A # are characterized by their ability to bind to phospholipid interfaces as the presumed first step in their biological action.…”

Modulation of prostaglandin E2 synthesis in rabbit synoviocytes

“…If phospholipase A2 was solely responsible for PGE2 synthesis, then one might expect intracellular C20:4 levels to remain elevated, rather than to decline toward control levels as was observed in this study. These experiments provide indirect evidence of an apparent increase in cyclooxygenase activity, which may be dependent on new enzyme synthesis, since the increase can be inhibited by cycloheximide or actinomycin D. One must be cautious about conclusions based on the effects of cycloheximide or actinomycin D because, in some systems, toxicity has been noted (13). However, in synoviocytes in the studies described here, viability was not affected, as judged by trypan blue dye exclusion and lactate dehydrogenase release.…”