Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils.

Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2). However, platelet-activating factor-induced arachidonic acid release is inhibited by both PD 98059 and SB 203580. Stimulation by TNF-alpha increases cPLA2 phosphorylation, which is inhibited by SB 203580, but not PD 98059, suggesting a role for p38 MAP kinase. LPS increases cPLA2 phosphorylation and arachidonic acid release. However, neither of these actions is inhibited by either PD 98059 or SB 203580. PMA increases cPLA2 phosphorylation. This action is inhibited by PD 98059 but not SB 203580. Finally, FMLP increases cPLA2 phosphorylation and arachidonic acid release. Interestingly, while the FMLP-induced phosphorylation of cPLA2 is not affected by the inhibitors of the p38 MAP kinase or erk cascades, both inhibitors significantly decrease arachidonic acid release stimulated by FMLP. SB 203580 or PD 98059 has no inhibitory effects on the activity of coenzyme A-independent transacylase.

Tumour necrosis factor-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils.

The role of the newly identified p38 mitogen-activated protein kinase (MAP kinase) in terminally differentiated cells, such as human neutrophils, is totally unknown. In order to examine the possible role of this MAP kinase in the phosphorylation and activation of cytoplasmic phospholipase A2 (cPLA2), we tested the effect of the recently synthesized inhibitor of p38 MAP kinase, SB 203580, on the phosphorylation and activation of both p38 MAP kinase and cPLA2. We found that while tumour necrosis factor-alpha (TNF-alpha)-stimulated tyrosine phosphorylation of p38 MAP kinase is affected only slightly by SB 203580, its stimulated kinase activity is greatly reduced in human neutrophils in suspension treated with this inhibitor. Furthermore, the TNF-alpha-stimulated phosphorylation and activation of cPLA2 are completely abolished in cells treated with SB 203580. Based on these data, it is reasonable to conclude that an SB 203580-sensitive kinase, or kinases and/or phosphatases, are involved in the phosphorylation and activation of cPLA2 in intact human neutrophils in suspension stimulated by TNF-alpha. The possible role of the p38 MAP kinase cascade in the phosphorylation and activation of cPLA2 is discussed.

Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists.

The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine-coupled receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-alpha). The effect of low concentrations of PAF is greatly increased in cells pretreated with LPS. The tyrosine phosphorylation of the p38 MAP kinase is not restricted to stimuli that mediate their actions through membrane-associated receptors, but it can be affected by agents that bypass membrane-associated receptors such as the protein translation blocker anisomycin. While anisomycin is known to increase the tyrosine phosphorylation of the 54 kDa SAPK (stress-activated protein kinase), this is the first report that shows that anisomycin also tyrosine phosphorylates the p38 MAP kinase. Cytokine receptors that increase the tyrosine phosphorylation and activation of the erk1 and erk2 MAP kinases have less effect on this p38 MAP kinase than those that do not affect the erk1 and erk2 MAP kinases. The possible role of the p38 MAP kinase in the phosphorylation of cytosolic phospholipase A2 is discussed.

Stimulation of intestinal Na+/H+ exchange by cell volume changes during fasting and refeeding in rats.

Intestinal cell water was studied in rats that have been fasted then refed, two conditions that are known to decrease and increase, respectively, proliferation of the intestinal epithelium. Cell water decreased (15%) during fasting and returned to normal with refeeding. The amiloride-sensitive sodium uptake, which estimates uptake through the Na+/H+ exchange was higher in the ileum than the jejunum, but the jejunal uptake, unlike the ileal, was significantly increased in fasted/refed rats than in normally fed or fasted ones. Osmotic shrinkage of intestinal cells followed by restitution of their cell volume stimulated the Na+/H+ exchange in all of the three groups of animals, but the increase was most prominent in the fasted and the fasted/refed groups. Also, shrinkage of cultured jejunal crypt cells (IEC-6) by a hypertonic solution increased intracellular alkalinization that was inhibited by amiloride. The results provide evidence for a relationship between the change in intestinal cell size, such as that which occurs during fasting/refeeding, and the activation of the Na+/H+ antiport system. This may represent one of the signals that initiates intestinal proliferation in the fasting/refeeding state.

Effect of lipopolysaccharide on mitogen-activated protein kinases and cytosolic phospholipase A2.

The addition of platelet-activating factor (PAF) to human neutrophils increases phosphorylation on tyrosine residues and stimulates the activity of p42erk2 mitogen-activated protein kinase (MAP kinase). This action is rapid and transient. In contrast, p42erk2, p44erk1 and the p40hera MAP kinase isoforms are all not tyrosine phosphorylated or activated in human neutrophils stimulated with low concentrations of lipopolysaccharide (LPS) in combination with serum. In spite of this, the PAF-induced tyrosine phosphorylation and activation of the p42erk2 MAP kinase are greatly potentiated in cells pretreated with LPS. More interestingly, although low concentrations of LPS do not affect MAP kinase isoforms in these cells, they cause the phosphorylation of cytosolic phospholipase A2 (cPLA2), as evidenced by a decrease in the electrophoretic mobility of the enzyme. In addition, this stimulus-induced upward shift in the mobility of the enzyme is not inhibited by the tyrosine kinase inhibitor, genistein. Furthermore, LPS increases the release of arachidonic acid in control and PAF-stimulated human neutrophils. These observations clearly show that cPLA2 can be phosphorylated and activated by kinases other than the currently known MAP kinases. It is proposed that there are MAP kinase-dependent and -independent mechanisms for the phosphorylation of cPLA2.

Cytoplasmic phospholipase A2 translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase.

The addition of the chemotactic peptide formylmethionylleucylphenylalanine (fMet-Leu-Phe) to human neutrophils pretreated with the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) results in a 10-fold enhanced activity of phospholipase A2, measured as the release of arachidonic acid. It is found that GM-CSF increases the tyrosine phosphorylation, enhances the activity of a mitogen-activated protein kinase, and greatly potentiates the fMet-Leu-Phe-induced tyrosine phosphorylation and enhanced activity of this kinase. Stimuli that increase the tyrosine phosphorylation, enhance the activity of the mitogen-activated protein kinase, and cause a rise in the intracellular concentration of free calcium increase the amount of phospholipase A2 associated with the plasma membrane. This increase corresponds to a decrease in the amount found in the cytosol. Whereas GM-CSF alone produces only a small increase in the amount of phospholipase A2 associated with the membrane, it potentiates greatly the fMet-Leu-Phe-induced increase. The total amount (whole cell) of phospholipase A2, as measured by immunoblotting using anti-phospholipase A2 antibody, does not change upon stimulation of human neutrophils with GM-CSF, fMet-Leu-Phe, or both. In addition, the band that corresponds to phospholipase A2 is shifted upward in membrane isolated from neutrophils stimulated with fMet-Leu-Phe, suggesting that the enzyme has been altered, possibly phosphorylated, though not on tyrosine residues. A working hypothesis is presented. Briefly, stimulation of human neutrophils with GM-CSF, in the absence of an additional stimulus, increases the tyrosine phosphorylation and activation of a mitogen-activated protein kinase, which in turn phosphorylates and activates cytoplasmic phospholipase A2. In the presence of an increased intracellular concentration of free calcium the phospholipase A2 is translocated to the plasma membrane where its substrate is located. GM-CSF also potentiates greatly the fMet-Leu-Phe-induced tyrosine phosphorylation and activation of a mitogen-activated protein kinase and, since fMet-Leu-Phe causes an intracellular calcium rise, the amount of the phospholipase A2 that is associated with the membrane fraction.

Pentoxifylline and CD14 antibody additively inhibit priming of polymorphonuclear leukocytes for enhanced release of superoxide by lipopolysaccharide: possible mechanism of these actions.

Lipopolysaccharide (LPS) primes human polymorphonuclear leukocytes (PMN) for enhanced O2- production in response to stimulation by N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe). Serum factor is essential for priming at lower concentrations of LPS. Complexes of LPS and LPS-binding protein are recognized by CD14 on PMN. We investigated the effects of a monoclonal antibody against CD14 (MY4) and of pentoxifylline (POF), a membrane fluidizer, alone and in combination, on LPS-LPS-binding protein activation of phospholipase D evidenced by increased phosphatidic acid formation. Phosphatidic acid formation and O2- production were inhibited by MY4 and POF. Our results suggest that the actions of these agents occur at an early step in the excitation-response sequence. In the absence of a second stimulus, LPS plus serum caused an increase in the amount of Gi alpha 2 associated with the membrane via CD14. POF, however, had no effect on Gi alpha 2 in the membrane. POF alone significantly changed the affinity (KD) of the fMet-Leu-Phe receptor of PMN (from 25.2 +/- 4.5 nM to 15.2 +/- 2.4 nM [P < 0.01; n = 4]) at 37 degrees C. The differences between the sites of action of MY4 and POF may lead to cooperation by these agents for inhibition of priming by LPS plus serum for enhanced O2- production. Clinical use of the antibody and POF may diminish tissue damage caused by PMN in clinical endotoxic shock.

Phorbol ester inhibits granulocyte-macrophage colony-stimulating factor binding and tyrosine phosphorylation.

Exposure of human polymorphonuclear neutrophils to phorbol 12-myristate 13-acetate (PMA) results in a 70-75% reduction in the specific binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) to its receptors. The PMA-induced reduction in 125I-GM-CSF binding is due to a decrease in the number of available GM-CSF receptors, as derived from Scatchard analysis of the binding data. On the other hand, the phorbol ester 4-alpha-phorbol 12,13-didecanoate (4 alpha-PDD) fails to affect 125I-GM-CSF binding. PMA promotes phosphorylation on tyrosine residues of several proteins, as demonstrated by Western blotting analysis using antiphosphotyrosine antibodies. The molecular masses of those proteins are 41, 55, 66, 78, 85, 104, and 115 kDa. GM-CSF increases the levels of the tyrosine phosphorylation of several proteins, the majority of which have similar Mr to those found in PMA-stimulated neutrophils. This increase, on all but the 41-kDa protein, is partially prevented by treatment of the cells with PMA. The inhibition by PMA of GM-CSF binding to its receptors and its phosphorylated effects is partially prevented by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and, to a greater extent, by staurosporine. It is suggested that PMA, through the activation of protein kinase C, interrupts the excitation-response sequence initiated by GM-CSF, which includes tyrosine phosphorylation, and that the earliest altered step is the binding of GM-CSF to its receptor.

Modulation of leukotriene B4 and platelet-activating factor binding to neutrophils.

Preincubation of human neutrophils with the human hormone granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibits the specific binding of leukotriene B4 ([3H]LTB4) but not the nonmetabolizable bioactive platelet-activating factor ([3H]C-PAF) to intact cells. This inhibition requires that the GM-CSF interacts with intact cells. The action of GM-CSF is not prevented by pertussis toxin. Moreover, the rise in calcium produced by LTB4 but not by PAF is also inhibited in human neutrophils pretreated with GM-CSF. Interestingly, neither the inhibitory action of GM-CSF on [3H]LTB4 binding or LTB4-induced calcium rise nor the potentiation of superoxide production by GM-CSF is reduced by inhibitors of arachidonic acid metabolism by the lipoxygenase pathway. In contrast, preincubation of human neutrophils with either the chemotactic factor formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), inhibits the binding of both [3H]LTB4 and [3H]C-PAF to intact cells. The inhibitory actions of GM-CSF, PMA, and fMet-Leu-Phe require that they interact with the intact cells; their actions cannot be reproduced in plasma membrane preparations. The effects of both GM-CSF and fMet-Leu-Phe cannot be prevented by the protein kinase C inhibitor staurosporine. The mechanisms of fMet-Leu-Phe and GM-CSF actions are probably not mediated through the release of LTB4 by the cells. Interestingly, this new action, unlike other reported effects of GM-CSF, is not mediated through a pertussis toxin-sensitive G protein (Gi alpha 2). This indicates that not all GM-CSF receptors are coupled to Gi alpha 2.

Calcium is necessary but not sufficient for the platelet-activating factor release in human neutrophils stimulated by physiological stimuli. Role of G-proteins.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; PAF) enhances the release of newly synthesized PAF as measured by [3H]acetate incorporation into PAF in human neutrophils. The response was dose-dependent, rapid, transient, and inhibitable by the PAF antagonist BN-52021. The non-metabolizable bioactive PAF analogue (C-PAF) but not lyso-PAF enhances the release of newly synthesized PAF. Newly synthesized PAF was also released after stimulation of these cells with fMet-Leu-Phe. The human granulocyte-macrophage colony-stimulating factor potentiates the stimulated release of PAF. The intracellular calcium chelator BAPTA inhibits the rise of [Ca2+]i and the release of PAF but not the Na+/H+ antiport activity. PAF release, but not the rise in the intracellular concentration of free calcium, was inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The release of PAF in pertussis toxin-treated cells was also inhibited in cells stimulated with fMet-Leu-Phe or opsonized zymosan. These results suggest that functional pertussis toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for PAF release produced by physiological stimuli. It appears that PAF release requires a coordinated action of receptor-coupled G-proteins, calcium, and other parameters.

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 of 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 molecular 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 pertussis toxin. Several important conclusion can be drawn from these data. 1) GM-CSF potentiates the rise in Ca2+i 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.

Phorbol 12-myristate 13-acetate inhibits binding of leukotriene B4 and platelet-activating factor and the responses they induce in neutrophils: site of action.

The addition of the platelet-activating factor (PAF) to neutrophils causes an increase in cytoskeletal actin, a rise in the intracellular concentration of free calcium, release of arachidonic acid, and the synthesis of PAF. The PAF synthesis in human neutrophils stimulated by PAF is greatly potentiated by the human granulocyte-macrophage colony-stimulating factor. Incubation of human neutrophils with the tumor copromoter phorbol 12-myristate 13-acetate (PMA) for 3 min prior to the addition of the stimulus inhibits all these responses produced by PAF. The inhibition is prevented when the cells are incubated with protein kinase C inhibitors such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine for 5 min prior to the addition of PMA. The rise in the intracellular concentration of free calcium in human neutrophils stimulated with leukotriene B4 is also inhibited by PMA, and this inhibition is prevented by protein kinase C inhibitors such as staurosporine. Unlike PMA, the inactive ester 4 alpha-phorbol 12,13-didecanoate has no inhibitory effect on the stimulated rise in the intracellular concentration of free calcium. The binding of either PAF or leukotriene B4 to intact cells is inhibited by PMA. The most important finding of the present studies is that PMA interferes with the binding of PAF and leukotriene B4 to their respective receptors. Whether PMA inhibits the binding of these lipid mediators by activating protein kinase C or by perturbing the membrane directly remains to be elucidated.

Granulocyte-macrophage colony-stimulating factor and human neutrophils: role of guanine nucleotide regulatory proteins.

The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to human neutrophils causes a rapid increase in the basal and fMet-Leu-Phe-stimulated Na+ influx and an increase in intracellular pH. The increase can be seen as early as 5 min after the addition of GM-CSF. Changes produced by GM-CSF are totally inhibited by amiloride and are significantly reduced in pertussis toxin-treated cells. The stimulation of the Na+/H+ exchange mechanism by GM-CSF inhibits further stimulation of this system with either fMet-Leu-Phe or phorbol 12-myristate 13-acetate. In addition, membrane preparations isolated from GM-CSF-treated neutrophils have higher basal and stimulated GTPase activities. The basal and the fMet-Leu-Phe- or platelet-activating factor-stimulated GTPase activities are reduced in pertussis toxin-treated cells. Cells pretreated with GM-CSF accumulate more radioactive phosphate than control cells, and this increase is diminished by pertussis toxin treatment. In addition, GM-CSF causes a rapid increase in the tyrosine phosphorylation levels of five proteins with molecular masses of 118 kDa, 92 kDa, 78 kDa, 54 kDa, and 40 kDa. These results clearly show that GM-CSF, on its own, can initiate several changes and that these changes are mediated in part by the pertussis toxin-sensitive guanine nucleotide regulatory protein.

Characterization of the membrane-associated GTPase activity: effects of chemotactic factors and toxins.

Membranes prepared from rabbit neutrophils exhibit GTPase activity which can be stimulated by the chemotactic factor fMet-Leu-Phe. The maximum contribution of the ATPase activities to the basal and the fMet-Leu-Phe-stimulated GTPase activities are less than 20% and 9%, respectively. The basal GTPase activity has a Vmax = 34.2 +/- 1.3 (pmol/mg protein, min) and a Km = 0.39 +/- 0.03 microM; and the fMet-Leu-Phe-stimulated has a Vmax = 52.3 +/- 2.5 (pmol/mg protein, min), and a Km = 0.29 +/- 0.02 microM. The GTPase activity can be stimulated by fMet-Leu-Phe and leukotriene B4. Unlike these two chemotactic factors, concanavalin A does not stimulate this GTPase activity. In addition, the rise in intracellular concentration of free calcium produced by concanavalin A is not inhibited by pertussis toxin treatment. Both the basal and stimulated GTPase activities are affected by pertussis toxin, cholera toxin and N-ethylmaleimide.

Arachidonic acid release in rabbit neutrophils.

[3H]Arachidonic acid is released after stimulation of rabbit neutrophils with fMet-Leu-Phe or platelet-activating factor (PAF). The release is rapid and dose-dependent, and is inhibited in phorbol 12-myristate 13-acetate (PMA)-treated rabbit neutrophils. The protein kinase C (PKC) inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine (H-7) prevents this inhibition. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. [3H]Arachidonic acid release, but not the rise in the concentration of intracellular Ca2+, is inhibited in pertussis-toxin-treated neutrophils stimulated with PAF. The diacylglycerol kinase inhibitor R59022 increases the concentration of diacylglycerol and potentiates [3H]arachidonic acid release in neutrophils stimulated with fMet-Leu-Phe. This potentiation is not inhibited by H-7. These results suggest several points. (1) A rise in the intracellular concentration of free Ca2+ is not sufficient for arachidonic acid release in rabbit neutrophils stimulated by physiological stimuli. (2) A functional pertussis-toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for arachidonic acid release produced by physiological stimuli. (3) Agents that stimulate PKC potentiate arachidonic acid release, and this potentiation is not inhibited by H-7. These agents produce their actions in part by direct membrane perturbation.

Actions of the protease inhibitor phenylmethylsulfonyl fluoride on neutrophil granule enzyme secretion and superoxide production induced by fMet-Leu-Phe and phorbol 12-myristate-13-acetate.

The protease inhibitor, phenylmethylsulfonyl fluoride inhibits granule enzyme release and, above 1 mM, superoxide production from rabbit peritoneal neutrophils induced by the chemotactic peptide, fMet-Leu-Phe. At concentrations below 1 mM, it enhances superoxide production. Superoxide generation stimulated by phorbol 12-myristate-13-acetate is increased by phenylmethylsulfonyl fluoride at all concentrations studied. Phenylmethylsulfonyl fluoride has no effect on the rise in intracellular calcium or the depolarization induced by fMet-Leu-Phe but does decrease the extent of repolarization and abolishes hyperpolarization. It depresses actin polymerization and abolishes cytoplasmic alkalinization caused by fMet-Leu-Phe. The increased phosphorylation induced by phorbol 12-myristate-13-acetate in four of the five proteins studied was not affected by phenylmethylsulfonyl fluoride, but the increased phosphorylation of the fifth, a 21-kD protein was enhanced. We conclude that phenylmethylsulfonyl fluoride acts on inhibitory and enhancing processes or steps induced by fMet-Leu-Phe which are subsequent to or independent of calcium mobilization and protein kinase C activity.

Diacylglycerol kinase inhibitor R59022 and stimulated neutrophil responses.

The generation of phosphatidic acid in neutrophils stimulated by the chemotactic factor formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) is inhibited by the diacylglycerol kinase inhibitor R59022. Superoxide generation produced by fMet-Leu-Phe, leukotriene B4, platelet-activating factor, or phorbol 12-myristate 13-acetate can be greatly increased in neutrophils pretreated with R59022. The potentiation occurs in the presence or absence of cytochalasin B and is evident in the absence of extracellular calcium. In addition, where the superoxide generated by fMet-Leu-Phe is not inhibited by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), the increase by R59022 is diminished by this compound. Unlike cytochalasin B, R59022 does not affect the increase in cytoskeletal actin produced by fMet-Leu-Phe or platelet-activating factor nor does it decrease the basal level. Furthermore, the basal intracellular concentration of free calcium, but not the rise produced by fMet-Leu-Phe or platelet-activating factor, is elevated by R59022. The data presented here suggest that the potentiation by R59022 of the oxidative burst is most likely mediated through protein kinase C.

Effect of cholera toxin on histamine release from bone marrow-derived mouse mast cells.

Bone marrow-derived mouse mast cells were sensitized with monoclonal mouse IgE antibody and treated with cholera toxin (CT), which ADP-ribosylated the alpha-subunit of the stimulatory guanine nucleotide-binding regulatory protein Gs, prior to challenge with either antigen or thrombin. The CT treatment increased intracellular cAMP levels, but neither enhanced nor inhibited antigen-induced histamine release or arachidonate release. The same treatment of the sensitized bone marrow-derived mouse mast cells with CT markedly enhanced thrombin-induced histamine release without affecting arachidonate release. The CT treatment failed to affect antigen-induced and thrombin-induced generation of inositol trisphosphate and of diacylglycerol or mobilization of intracellular Ca2+. The results indicate that Gs in bone marrow-derived mouse mast cells is not involved in the transduction of the antigen-induced or thrombin-induced triggering signal to phospholipase C, which initiates the enhancement of phosphatidylinositol turnover. The enhancement of thrombin-induced histamine release by CT treatment with the observations that thrombin-induced histamine release was inhibited by pretreatment of the cells with pertussis toxin suggest that the involvement of a guanine nucleotide-binding regulatory protein in thrombin-induced biochemical events is an event distal to Ca2+ mobilization.

Intracellular calcium rise produced by platelet-activating factor is deactivated by fMet-Leu-Phe and this requires uninterrupted activation sequence: role of protein kinase C.

Stimulation of the neutrophils with fMet-Leu-Phe inhibits the rise in intracellular concentration of free calcium produced by the subsequent addition of platelet-activating factor. This deactivation is not observed in pertussis toxin treated cells. In addition, preincubation of the cells with the protein kinase C activator phorbol 12-myristate 13-acetate for three minutes abolishes completely the rise in calcium produced by platelet-activating factor. This inhibition is prevented by the addition of the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine prior to the addition of the phorbol ester. Phorbol 12-myristate 13-acetate, at a concentration that does not produce significant inhibition, accelerates the rate of calcium removal from the cytoplasm, and this is abolished by the protein kinase C inhibitor. In contrast, the deactivation by fMet-Leu-Phe is not prevented by the protein kinase C inhibitor. The results presented here suggest that the protein kinase C system may regulate the opening by platelet-activating factor of possible plasma membrane associated pertussis toxin independent calcium channels and/or the binding of platelet-activating factor to the receptors. In addition, protein kinase C activation increases the rates of the calcium efflux pump and/or calcium sequestering by intracellular organelles. The most simple and straightforward explanation of the observed deactivation by fMet-Leu-Phe is that the addition of fMet-Leu-Phe to neutrophils stimulates the production of platelet-activating factor which then binds to and deactivates the receptors.