Leukotriene B4 mobilizes calcium without the breakdown of polyphosphoinositides and the production of phosphatidic acid in rabbit neutrophils.

Abstract

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 ...

Human polymorphonuclear neutrophil activation with arachidonic acid

“…Further, several of the aforementioned cis-unsaturated fatty acids have been found to stimulate Ca2+ release from the sacroplasmic reticulum, an intracellular Ca2+ storage site, in skeletal muscle (Cheah, 1981). Consistent with our data is the observation that AA increases the [Ca2+]i in human and rabbit (Volpi et al, 1984) PMNs and degranulation by rabbit PMNs (Naccache et al, 1979 (Kajikawa et al, 1983;Nishizuka, 1984) and our data show AA to stimulate PMN PK-C at concentrations that induce degranulation. In addition, y-linolenic, linoleic and oleic acid have also been demonstrated to activate human PMN PK-C (McPhail et al, 1984) and their activating potencies correlate with their capacities to induced granule exocytosis from PMNs.…”

“…The possibility that AA-induced Ca2+ mobilization was mediated by phospholipase C-catalysed hydrolysis of phosphatidylinositol 4,5-bisphosphate with the release of inositol-1,4,5-trisphosphate would appear to be negated by the report that AA has no effect on phosphatidylinositol breakdown in PMNs (Volpi et al, 1984). However, AA could facilitate Ca2+ mobilization via a direct effect on the intracellular Ca2" storage site(s).…”

Prostaglandins E1 and E2 enhance the stimulation of superoxide release by 1‐oleoyl‐2‐acetylglycerol from human neutrophils

“…Stimulation of neutrophils with fMet-Leu-Phe has been reported to result in an increase of DAG in one study [25] but in another [26] it has been suggested that this may not occur. If this latter statement is correct, an explanation other than the one proposed here would be required for the PGEi effect on 0; generation by neutrophils stimulated with this peptide.…”

Clostridium difficile toxin A stimulates intracellular calcium release and chemotactic response in human granulocytes.

“…Chemotactic factors bind to membrane receptors on granulocytes, eliciting a wide range of responses including membrane depolarization (7), release of intracellular calcium (8), inositol phospholipid turnover (9), and protein phosphorylation (10). These signals in turn activate a full range ofgranulocyte responses including chemotaxis, degranulation, and oxygen radical production.…”