P.H. Naccache

Phorbol 12-myristate 13-acetate activates rabbit neutrophils without an apparent rise in the level of intracellular free calcium

The addition of low concentrations of phorbol 12-myristate 13-acetate to rabbit neutrophils induces cell aggregation, degranulation, increased oxygen consumption and an increase in the amount of actin associated with the cytoskeleton without a rise in the level of intracellular free calcium as measured using the fluorescent probe quin-2. The ability of phorbol 12-myristate 13-acetate to initiate neutrophil responses similar to those produced by the chemotactic factor without causing a rise in the level of intracellular free calcium suggests two possibilities; that there is a second messenger in addition to calcium or that it activates the cells at a point distal to calcium mobilization. The possible role of diacylglycerol in neutrophil activation is discussed.

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.

Chemotactic factor causes rapid decreases in phosphatidylinositol,4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils

Stimulation of rabbit neutrophils prelabeled with 32P by the synthetic chemotactic peptide f-Met-Leu-Phe induces a rapid decrease in the radioactivity in both phosphatidylinositol, 4,5 bis phosphate and phosphatidylinositol 4-monophosphate. The mean +/- standard error of the mean values of the maximum decrease in phosphatidylinositol, 4,5 bis phosphate occurred at 10 seconds following stimulation and is equal to 19 +/- 3% of the control value. The corresponding value for phosphatidylinositol 4-monophosphate occurred at 60 seconds following stimulation and is equal to 37 +/- 7% of the control value. On the other hand, the radioactivity in phosphatidic acid and lysophospholipids increased continuously with time following stimulation. The relationship of these changes to calcium release and neutrophil activation is discussed.

Specific modulation of the intracellular pH of rabbit neutrophils by chemotactic factors

Abstract We have monitored the intracellular pH of rabbit neutrophils by following the distribution of the weak acid, 5,5-Dimethyloxazolidine-2, 4-dione. The synthetic formyl-methionyl chemotactic factors were found to induce complex and specific changes in the intracellular pH of the neutrophils, a rapid drop followed by a slower and more sustained rise. These effects are receptor mediated. The relationship of these events to the physiology of the neutrophils is discussed.

Arachidonic acid induced degranulation of rabbit peritoneal neutrophils.

Abstract We have found that arachidonic acid rapidly and selectively induces the release of lysosomal enzymes from cytochalasin B treated rabbit peritoneal neutrophils. 5, 8, 11, 14-eicosatetraynoic acid inhibits the arachidonate induced release with an apparent K D of 1.5 × 10 −6 M. 5,8,11,14-eicosatetraynoic acid (2.5 × 10 −5 M also inhibits the chemotactic factors and the A23187 induced release in the presence of cytochalasin B but does not affect the degranulation induced by A23187 alone. These observations strongly suggest a role for arachidonate metabolites in rabbit neutrophil physiology.

Calmodulin inhibitors block neutrophil degranulation at a step distal from the mobilization of calcium

Summary Two calmodulin inhibitors, trifluoroperazine and N-(6-aminohexyl-5-chloro-1-naphtalene sulfonamide, were found to be potent inhibitors of the chemotactic factor plus cytochalasin B induced lysosomal enzyme release from rabbit peritoneal neutrophils. On the other hand, these inhibitors did not affect the chemotactic factor induced intracellular calcium redistribution and membrane permeability increase. These results strongly suggest that calmodulin is involved in the expression of the various neutrophil functions. In addition, these compounds provide a unique experimental tool for the dissection of the excitation-secretion coupling sequence in neutrophils and other secretory cells.

Pertussis but not cholera toxin inhibits the stimulated increase in actin association with the cytoskeleton in rabbit neutrophils: Role of the “G proteins” in stimulus-response coupling

Treatment of rabbit neutrophils with pertussis toxin, but not cholera toxin, inhibits the increases produced by formylmethionyl-leucyl-phenylalanine, leukotriene B4 and the calcium ionophore A23187 in the amounts of actin associated with the cytoskeletons. The increase in the cytoskeletal actin produced by phorbol 12-myristate, 13-acetate on the other hand is not affected by pertussis toxin. Incubation of the neutrophils with cholera toxin, unlike pertussis toxin, did not inhibit the fMet-Leu-Phe induced rise in the intracellular concentration of free calcium, and caused only a shift to the right of the dose-response curve of N-acetyl-beta-glucosaminidase release. This shift was more marked in the presence of 1-methyl-3-isobutylxanthine. In addition, the stimulated breakdown of phosphatidylinositol 4,5 bis-phosphate was inhibited by pertussis toxin. These results suggest that pertussis toxin acts at an early step in the signal transduction and does not affect the sequence of reactions initiated by the activation of the protein kinase C. Furthermore, the guanine nucleotide regulatory protein Gi, but not Gs, is closely involved in signal transduction in these cells.

Arachidonate metabolite(s) increase the permeability of the plasma membrane of the neutrophils to calcium

Abstract The relationship between arachidonate metabolism and stimulated calcium fluxes in rabbit peritoneal neutrophils has been investigated. The addition of arachidonate to the neutrophils was found to cause a rapid and significant increase in the permeability of the plasma membrane to calcium. This effect is specific to calcium, concentration dependent and sensitive to inhibitors of the lipoxygenase mediated metabolic pathway(s). These results strongly suggest that arachidonate metabolites are directly involved in the mechanisms underlying calcium gating in the neutrophils.

Unique inhibitory profile of platelet activating factor induced calcium mobilization, polyphosphoinositide turnover and granule enzyme secretion in rabbit neutrophils towards pertussis toxin and phorbol ester

Platelet activating factor has been found to increase the intracellular level of free calcium (as monitored by the fluorescent calcium indicator quin-2) and to stimulate the turnover of the polyphosphoinositides in rabbit neutrophils. Calcium mobilization induced by platelet activating factor, in contrast to previous reports with chemotactic factors, is unaffected by pertussis toxin; on the other hand, stimulated polyphosphoinositol hydrolysis and granule enzyme secretion are potently antagonized under the same conditions. The calcium, as well as the secretory responses to the lipid mediator are largely dependent on the presence of extracellular calcium. Internal contributions to the quin-2 signal are only detectable at relatively high concentrations of platelet activating factor. Calcium mobilization and secretion stimulated by platelet activating factor are inhibited following a short incubation with phorbol 12-myristate 13-acetate. These results are discussed in terms of the possibility that platelet activating factor activates neutrophils via dual pathways, the first involving direct interaction with phorbol ester inhibitable calcium channels and the other the stimulation in a manner dependent on a guanine nucleotide binding protein of the phospholipase C specific for polyphosphoinositides.

Phorbol 12-myristate, 13-acetate potentiates the action of the calcium ionophore in stimulating arachidonic acid release and production of phosphatidic acid in rabbit neutrophils

The addition of the tumor-promoting phorbol 12-myristate, 13-acetate to rabbit neutrophils greatly potentiates the effect of the calcium ionophore A23187 on [3H]-arachidonic acid release and [32P]-phosphatidic acid generation. At 5 X 10(-8) M A23187, the addition of 20 ng/ml PMA potentiates the action of the ionophore on [3H]-arachidonic acid release by 5-fold. At 5 X 10(-7) M A23187, PMA enhances [32P]-phosphatidic acid production by 1.5-fold. Incubation of the neutrophils with 5 X 10(-7) M ionophore for two minutes causes a significant increase in the [32P] phosphatidic acid production but does not affect the levels of [32P]-phosphatidylinositol or [32P]-phosphatidylinositol 4,5 bis-phosphate. In addition, increasing the sodium chloride concentrations in the suspending medium causes an increase in the level of phosphatidylinositol 4,5 bis-phosphate. These results suggest that the phorbol ester either acting directly or through the activation of protein kinase C modulates significantly the activities of the various forms of phospholipases, particularly A2, and/or increases the availability or amounts of their substrates.