Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.

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. The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine . On the other hand, the addition of pertussis toxin or hyperosmolarity has small, if any, effect on the rise in intracellular calcium produced by S SME ofthe neutrophil responses that are activated by the chemotactic factor formylMethionyl-Leucyl-Phenylalanine (Met-Leu-Phe)' such as cell motility, shape change, and the projection of pseudopodia or ruffles depend on the mechanical displacement of part of the cell, or of the whole cell. The cellular contractile apparatus, of which actin and myosin are the major components, is closely involved in these responses, and an understanding of neutrophil activation requires a detailed knowledge ofthe organization ofthese proteins before and after stimulation . Actin filaments in neutrophils are considerably more labile than their counterpart in muscle, and large pools of depolymerized actin are usually found in resting cells (Korn, 1982;Pollard, 1975 ;Southwick and Stossel, 1983).Recently, it has been shown that the addition of fMet-LeuPhe to neutrophils causes a rapid polymerization of actin (Roa and Varani, 1982;White et al., 1983a; Fechheimer and Zigmond, 1983; Yassin et al., 1985). The signals that initiate actin polymerization are not known, but are important to determine because of the central role of actin in neutrophil functions (Southwick and Stossel, 1983; Yassin et al., 1985;Fox et al ., 1984). Under conditions similar to those under which actin gets polymerized, chemotactic factors also in-'Abbreviations used in this paper: fMet-Leu-Phe, formylMethionyl-LeucylPhenylalanine ; IP3, inositol, 1,4,5-trisphosphate; PAF, platelet-activating factor; PIP2, phosphatidylinositol 4,5-bis-phosphate ; PMA, phorbol 12-myristate, 13-acetate.C The Rockefeller University Press, 0021-9525/86/04/1459/05 $1 .00 The Journal of Cell Biology, Volume 102, April 1986 1459-1463 A23187 . While quinacrine does not affect the fMetLeu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus . The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed . crease the hydrolysis of phosphatidylinositol 4,5-bis-phosphate and causes a rise in the level ofintracellular concentration of free calcium (White et al., 1983;Roa and Varani, 1982;Volpi et al., 1983; White et al., 198...

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 ...

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