The addition of fMet-Leu-Phe, leukotriene B4, or arachidonic acid to rabbit neutrophils causes a rise in the level of intracellular free calcium as measured by the fluorescent dye quin-2. The calcium response is rapid and dosedependent with an ED50 of 0.12 ± 0.05 nM for leukotriene B4, 0.20 ± 0.02 nM for fMet-Leu-Phe, and 320 ± 30 nM for arachidonic acid. However, unlike fMet-Leu-Phe, leukotriene B4 at concentrations up to 70 nM does not cause a significant breakdown of any of the phosphoinositides or the generation of phosphatidic acid, arachidonic acid, or 1,2-diacylglycerol. The addition of arachidonic acid causes little (<20%) production of phosphatidic acid. Furthermore, the dose-response curve of the generation of phosphatidic acid by fMet-Leu-Phe is shifted to the right when compared with that for calcium mobilization. The results reported here indicate that, although similar in many respects, there are important qualitative differences between the mechanisms of action of these two chemotactic factors. They also show that calcium mobilization by leukotriene B4 is not mediated by the breakdown of any of the phosphoinositides, and calcium mobilization by leukotriene B4, arachidonic acid, and possibly fMet-Leu-Phe is not mediated by the generation of phosphatidic acid.The activation of neutrophils by chemotactic factors and other stimuli is thought to be mediated by a rise in the level of intracellular calcium and/or activation of the protein kinase C *ystem (1-10). While some of these stimuli produce their effects by eliciting a rise in the level of intracellular calcium, others produce their effects by activating the protein kinase C system directly. Recently, it has been suggested that there are other second messengers (1,2-diacylglyceride) in addition to calcium ion that may be involved in neutrophil and platelet activation (11,12). It has been demonstrated that occupancy of receptors by soluble stimuli activates phospholipase C. This activation generates inositol 1,4,5-tris(phosphate) and 1,2-diacylglycerol from the breakdown of phosphatidylinositol 4,5-bis(phosphate). Some of the 1,2-diacylglycerol is converted to phosphatidic acid, and arachidonic acid may be released from either of these products.It has been hypothesized that calcium ion is released through the action of the generated inositol 1,4,5-tris(phosphate) (13)(14)(15)(16)(17). This hypothesis holds that the initial lipolytic response to stimulation does not require a rise above the basal level of intracellular free calcium. Thus, the breakdown of phosphatidylinositol 4,5-bis(phosphate) is viewed as directly receptor-linked and ultimately responsible in turn for the mobilization of calcium necessary for cell activation. Chemotactic stimuli, such as the synthetic peptide fMet-Leu- (19). [3H]Arachidonic acid (final concentration, 5 uCi/ml; 1 Ci = 37 GBq) was sampled into a roundbottom flask, the ethanol was evaporated with a stream of nitrogen, and immediately a known volume of a cell suspension (108 cells per ml) was added. The cell ...

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...

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The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.