T.F.P. Molski

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.

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.

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.

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.

Biochemical events associated with the stimulation of rabbit neutrophils by platelet-activating factor.

The functional and biochemical responses evoked by the addition of platelet‐activating factor (PAF) to a suspension of rabbit neutrophils have been characterized in an effort to define the mode of action of this lipid mediator. PAF was found to elicit a secretory response and to stimulate a rapid breakdown of the polyphosphoinositides, an increase in the cytoplasmic level of free calcium (as monitored by quin2), a decrease in the fluorescence of cell‐associated chlortetracycline, an enhanced activity of the sodium/hydrogen antiport, a transient depolarization, and an increase in the level of cytoskeletal actin. The quin2 response to PAF was found to be detectable at concentrations as low as 0.01 nM, to be very dependent on the presence of extracellular calcium, and to be sensitive to inhibition by phorbol esters. On the other hand, the increase in free calcium induced by PAF in the presence of extracellular calcium was essentially unaffected by pertussis toxin. PAF‐induced neutrophil degranulation was similarly extracellular calcium dependent and phorbol ester sensitive. The secretory activity of PAF was evident only at concentrations in excess of 1 nM. All of the other effects of PAF were found to be independent of the presence of external calcium and to be demonstrable only at concentrations larger than 1 nM. In addition, all neutrophil responses to PAF (with the above noted exception of quin2) were potently inhibited by pertussis toxin. These results are interpreted in terms of the possible existence of two functionally distinct populations of receptors. The occupation of one set (of apparent high affinity) induces an increase in permeability to calcium in a phorbol‐ester‐, but not pertussis‐toxin‐, sensitive manner. The activation of the other set of receptors at higher concentrations of PAF stimulates the polyphosphoinositide‐specific phospholipase C and induces the attendant biochemical responses. These latter responses appear to be mediated by a guanine‐nucleotide‐binding regulatory protein.

Treatment of rabbit neutrophils with phorbol esters results in increased ADP-ribosylation catalyzed by pertussis toxin and inhibition of the GTPase stimulated by fMet-Leu-Phe

The effects of pretreatment of rabbit neutrophils with phorbol 12‐myristate 13‐acetate on the ability of pertussis toxin to catalyze ADP‐ribosylation and of fMet‐Leu‐Phe to activate a high‐affinity GTPase in these cell homogenates were examined. The addition of phorbol 12‐myristate 13‐acetate, but not 4α‐phorbol 12,13‐didecanoate, to intact cells was found to stimulate by more than 100% the pertussis toxin‐dependent ribosylation of a 41 kDa protein (either the α‐subunit of the ‘inhibitory’ guanine nucleotide‐binding protein Ni or a closely analogous protein) and to inhibit by more than 60% the activation by fMet‐Leu‐Phe of the GTPase of the neutrophil homogenates. The addition of fMet‐Leu‐Phe to intact cells increases the ADPribosylation catalyzed by pertussis toxin of the 41 kDa protein. On the other hand, the exposure of neutrophil homogenates to fMet‐Leu‐Phe results in a decreased level of ADP‐ribosylation. This decreased ribosylation reflects a dissociation of the GTP‐binding protein oligomer that is not followed by association, possibly because of the release of the α‐subunit into the suspending media. The implications of these results for the understanding of the mechanism of inhibition of cell responsiveness by phorbol esters and the heterologous desensitization phenomenon are discussed. Prominent among these are the possibilities that (i) the rate of dissociation of the Ni oligomer is affected by the degree of its phosphorylation by protein kinase C, and/or (ii) the dissociated phosphorylated α‐subunit (the 41 kDa protein) is functionally less active than its dephosphorylated couterpart.

Effect of granulocyte-macrophage colony-stimulating factor on superoxide production in cytoplasts and intact human neutrophils: role of protein kinase and G-proteins.

Granulocyte‐macrophage colony‐stimulating factor, GM‐CSF, potentiates superoxide generation produced by human neutrophils stimulated with fMet‐Leu‐Phe and platelet‐activating factor, PAF, but not by phorbol 12‐myristate 13‐acetate (PMA) or opsonized zymosan. The potentiation is greatest in fMet‐Leu‐Phe‐stimulated cells. This indicates that the actions of only certain receptors are potentiated by GM‐CSF. Incubation of the cells with the protein kinase inhibitor H‐7 or with the protein synthesis inhibitor cyclohexamide before the addition of GM‐CSF does not affect the observed potentiation. The rationales behind these studies are to examine the roles of protein kinase C and protein synthesis in the action of GM‐CSF. The data suggest that neither protein kinase C nor protein synthesis is necessary for GM‐CSF action. On the other hand, no potentiation can be seen in the presence of cytochalasin B. Unlike intact cells, GM‐CSF does not enhance superoxide production by cytoplasts stimulated with fMet‐Leu‐Phe. The rationale behind the use of cytoplasts is to examine the role of granules and/or nucleus in GM‐CSF action, and the data indicate that one or more of these two components is necessary for the priming effect of GM‐CSF. The amount of actin associated with the cytoskeleton under control or fMet‐Leu‐Phe‐stimulated condition is the same in normal and GM‐CSF‐treated human neutrophils. Botulinum D toxin ADP‐ribosylates a protein with a moleuclar weight of 22 kDa This ribosylation is reduced in homogenates obtained from cells pretreated with botulinum D toxin or GM‐CSF. Botulinum D toxin does not affect the basal or the fMet‐Leu‐Phe‐induced rise in the intracellular concentration of free calcium in human neutrophils. GM‐CSF also increases the rise in intracellular concentration of free calcium in human neutrophils stimulated with PAF or fMet‐Leu‐Phe. The increases are inhibited by petussis toxin, Several important conclusion can be drawn from these data. 1) GM‐CSF potentiates the rise in Ca2+, produced by PAF and fMet‐Leu‐Phe, and these potentiations are inhibited in pertussis‐toxin‐treated cells. 2) GM‐CSF does not prime cytoplasts to stimulation by fMet‐Leu‐Phe. This suggests that the granules and/or nucleus are necessary for the priming action. 3) The priming by GM‐CSF is not mediated by the H‐7‐sensitive protein kinase C, botulinum D‐sensitive G‐protein, or protein synthesis.

Dissociation of the 47-kilodalton protein phosphorylation from degranulation and superoxide production in neutrophils.

The aim for the present studies is to examine the relationship between the phosphorylation of the 47‐kDa protein and some neutrophil responses such as degranulation, the synergistic effect of PMA on calcium ionophore‐induced degranulation, superoxide generation, and the priming of the oxidative burst produced by the chemotactic factor fMet‐Leu‐Phe and phorbol 12‐myristate 13‐acetate (PMA).