Neutrophil activation by surface bound IgG is via a pertussis toxin insensitive G protein

Pre-treatment of neutrophils with either pertussis or cholera toxins does not inhibit neutrophil activation by surface bound IgG. In contrast, pretreatment with the phorbol ester, phorbol myristate acetate, results in a dose dependent inhibition of degranulation by surface bound IgG. This inhibition is similar to that seen with soluble ligands where it is thought to be due to interference with the interaction of an activated guanine nucleotide binding protein with phospholipase C (J. Biol. Chem.,262,6121,1987). More directly, GTP binding and GTPase activity are enhanced when human neutrophil membranes are incubated in wells containing surface bound IgG. Neither of these G protein functions were inhibited when membranes were prepared in the presence of pertussis toxin, suggesting that neutrophil activation by surface bound IgG proceeds by a mechanism that involves a pertussis toxin insensitive G protein.     

A protein kinase C inhibitor, staurosporine, activates phospholipase D via a pertussis toxin-sensitive GTP-binding protein in rabbit peritoneal neutrophils.

In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (> 1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils.     

Different sensitivity to phorbol esters and pertussis toxin of bombesin- and platelet-derived growth factor-induced, phospholipase C-mediated hydrolysis of phosphoinositides in NIH/3T3 cells

Incubation of the serum-deprived cultures of NIH/3T3 cells with bombesin or platelet-derived growth factor (PDGF) induced the phospholipase C-mediated hydrolysis of phosphoinositides. Protein kinase C-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) and pertussis toxin inhibited the bombesin-induced phospholipase C reactions. AlF4-, a direct activator of GTP-binding proteins (G proteins), also induced the phospholipase C reactions and TPA inhibited the AlF4- -induced reactions. These results suggest that a pertussis toxin-sensitive G protein is involved in the coupling of the bombesin receptor to the phospholipase C and that the coupling of the G protein to the phospholipase C is inhibited by protein kinase C. In contrast, neither TPA nor pertussis toxin inhibited the PDGF-induced phospholipase C reactions, indicating that a pertussis toxin-sensitive G protein is not involved in the coupling of the PDGF receptor to the phospholipase C and that this coupling is insensitive to protein kinase C. These results suggest that the regulatory mechanism of the PDGF receptor for the phospholipase C activation is different from that of the bombesin receptor.     

Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate.

The signal transduction mechanisms involved in the regulation of phagocytosis are largely unknown. We have recently shown that in neutrophils, when IgG-mediated phagocytosis is stimulated by formyl-methionyl-leucyl-phenyl-alanine (fMLP), the enhanced ingestion is dependent on the increase in [Ca2+]i which results from ligation of Fc receptors by the IgG-coated target (Rosales, C., and Brown, E. (1991) J. Immunol. 146, 3937-3944). Now, we have studied the mechanism by which this rise in [Ca2+]i occurs. Aggregated IgG, the monoclonal antibody 3G8 (which recognizes Fc receptor type III), and insoluble immune complexes caused an increase in [Ca2+]i. The rise in [Ca2+]i induced by Fc receptor ligation was resistant to pertussis toxin. In contrast, fMLP induced a rise in [Ca2+]i which was inhibited by pertussis toxin. fMLP-induced [Ca2+]i was accompanied by an accumulation of inositol 1,4,5-trisphosphate (IP3) which peaked by 15 s, and which was also abolished by pertussis toxin. IP3 accumulation after aggregated IgG, 3G8, or insoluble immune complexes was much less than after fMLP. Unlike [Ca2+]i rise induced by Fc receptor ligation, this small increase in IP3 was inhibited by pertussis toxin. These data demonstrated that the [Ca2+]i increase induced by Fc receptor ligation is not mediated by IP3. Immediate pretreatment of human polymorphonuclear neutrophils with optimal doses of fMLP also reduced subsequent increase in [Ca2+]i rise from thapsigargin, a sesquiterpene lactone tumor promoter that releases intracellular Ca2+ from IP3-sensitive stores without IP3 turnover. Similarly, to its effects on thapsigargin, fMLP inhibited the [Ca2+]i rise upon subsequent immune complex binding. Pretreatment of cells with immune complexes also prevented subsequent [Ca2+]i rise from thapsigargin and fMLP. These data demonstrate that IgG Fc receptor ligation and fMLP activation of human polymorphonuclear neutrophils use distinct signal transduction mechanisms to release Ca2+ from the same thapsigargin-sensitive intracellular pool. In contrast to fMLP, signal transduction for increased [Ca2+]i after Fc receptor stimulation does not involve a pertussis toxin-sensitive G protein, and is independent of IP3.     

Neutrophil activation by surface bound IgG: pertussis toxin insensitive activation

Surface bound IgG induces neutrophil degranulation and production of superoxide radicals by a mechanism that is not inhibited by either pertussis toxin or cholera toxin, whereas these functions induced by soluble mediators such as FMLP and soluble aggregates of IgG are profoundly inhibited by pertussis toxin. Interaction of neutrophils with surface bound IgG triggers the loss of 32P labeled PIP2 and PIP and the influx of extracellular calcium. Neither of these cellular events when induced by surface bound IgG is inhibited by pertussis toxin. These observations suggest that neutrophil activation induced by surface bound IgG proceeds along a pathway which is not regulated by proteins which are inhibited by either pertussis or cholera toxins.     

Stimulation of Primed Neutrophils by Soluble Immune Complexes

Soluble IgG-containing immune complexes are unable to initiate oxidant production in unprimed neutrophils. However, priming of the neutrophils with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or cytochalasin B prior to exposure to these complexes results in activation of a rapid and extensive secretion of reactive oxidants. In this study, we have investigated the ability of soluble immune complexes to: (1) induce oxidant generation; (2) bind to the cell surface; and (3) induce Ca²⁺transients in neutrophils primed by incubation with GM-CSF of cytochalasin B. Our findings give new insight into the molecular processes involved in the “priming” phenomenon.     

Pertussis toxin as a probe of neutrophil activation

In reviewing our own and other work, it is clear that pertussis toxin treatment of neutrophils causes a time- and concentration-dependent inhibition of granule enzyme secretion induced by formylmethionylleucylphenylalanine (fMet-Leu-Phe), C5a, leukotriene (LT) B4 and platelet-activating factor (PAF). Chemotaxis, O2- generation, aggregation, and arachidonic acid production induced by fMet-Leu-Phe are also inhibited by pertussis toxin. Granule enzyme release caused by A23187 or phorbol 12-myristate 13-acetate is not inhibited. The inhibition of neutrophil function correlates closely with the NAD-ribosylation of a 41,000-dalton protein in the neutrophil plasma membrane, presumably the GTP-binding regulatory protein Ni. Pertussis toxin treatment prevents or obtunds the increased influx of Ca2+ induced by fMet-Leu-phe and LTB4, but not that caused by stimulation of neutrophils with PAF. Pertussis toxin prevents the receptor-induced breakdown of polyphosphoinositides in intact neutrophils and isolated membrane and prevents or decreases the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. The hypothesis advanced by us and others is that pertussis toxin interacts with a GTP-binding regulatory protein identical or similar to Ni, which couples receptor-chemotactic factor interaction to phospholipase C activation. Inhibition of the activation prevents the production of IP3 and the resulting release of Ca2+ from intracellular stores and of 1,2-diacylglycerol and thus, the activation of protein kinase C. The lack of these two mediators is the immediate cause of the depression of neutrophil activation resulting from pertussis toxin. Some of the limitations and uncertainties of our present knowledge with respect to this hypothesis are discussed.     

Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblastoma N1E115 cells via both pertussis toxin sensitive and insensitive mechanisms

Neurotensin, bradykinin and somatostatin inhibited in a time- and concentration-dependent manner prostaglandin E1- or forskolin-stimulated cAMP production in neuroblastoma N1E115 cells. Cell treatment with 1 microgram/ml pertussis toxin for 6 hours reversed the inhibition elicited by peptides after short incubation periods (less than or equal to 1 min) but, in contrast, had no effect after longer incubation periods (greater than or equal to 3 min). Fluoroaluminate also inhibited prostaglandin E1-stimulated cAMP production in N1E115 cells, and this effect was not reversed by pertussis toxin. The 6 hour treatment with pertussis toxin was shown to be sufficient to ADP ribosylate virtually all of the 41 kD protein substrate corresponding to the alpha subunit of Gi. Protein kinase C activation with phorbol ester did not inhibit basal or stimulated cAMP production. Our data point to the existence of both pertussis toxin sensitive and insensitive mechanisms of neuropeptide-mediated inhibition of cAMP formation in N1E115 cells. The toxin insensitive response is not mediated by protein kinase C. The possibility is discussed that it results from the activation of a pertussis toxin insensitive G protein.     

Collagen-binding motif peptide, a cleavage product of osteopontin, stimulates human neutrophil chemotaxis via pertussis toxin-sensitive G protein-mediated signaling

The collagen-binding motif (CBM) peptide, a cleavage product of osteopontin (OPN), stimulated intracellular calcium increase in human neutrophils. CBM peptide-stimulated calcium was inhibited by pertussis toxin (PTX), suggesting the influence of PTX-sensitive G-proteins. In addition CBM peptide stimulated the chemotactic migration of human neutrophils and human monocytes. CBM peptide-induced neutrophil chemotaxis was completely inhibited by PTX, once again indicating the influence of Gi proteins. CBM peptide was also found to induce mitogen activated protein kinase activation. CBM peptide-induced neutrophil chemotaxis was mediated by p38 kinase as well as an extracellular signal-regulated protein kinase. Taken together, the results suggest that a cleavage product of OPN, CBM peptide, initiates immune responses by inducing neutrophil trafficking via certain PTX-sensitive cell surface receptors.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig periotoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig peritoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils

Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When [3H] AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of [3H]AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of [3H]AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate). These results suggest several points: 1) PAF stimulates human polymorphonuclear neutrophils to liberate AA mainly by the action of phospholipase A2; 2) Ca2+ mobilization alone is not sufficient to stimulate AA release, although Ca2+ is the important factor for phospholipase A2 activation; and 3) a pertussis toxin-sensitive GTP-binding protein may be implicated in activation of phospholipase A2.     

Calcium changes in immune complex-stimulated human neutrophils. Simultaneous measurement of receptor occupancy and activation reveals full population stimulus binding but subpopulation activation

Immune complexes (ICs) induce an initial transient increase in cytosolic intracellular calcium [( Ca2+]in) levels in human neutrophils (PMN). Changes in PMN [Ca2+]in were measured with the fluorescent calcium indicator Indo-1 ( [1-[2-amino-5-(6-carboxylindol-2-yl]-phenoxyl]-2-(2'-amino-5 '- methylphenoxy]ethane-N,N,N'N'-tetraacetic acid), at the level of individual cells by flow cytometry. Two kinds of immune complexes (ICs) were used in this study: an insoluble (IIC) and a more soluble less valent immune complex (SIC) with fewer available Fc receptor binding ends per molecule of SIC than IIC. Simultaneous binding and activation studies performed on the flow cytometer with fluoresceinated IIC or SIC demonstrated that a majority of the cells bound each stimulus uniformly. However, only an IC dose-dependent proportion of those IC-bound cells responded with an increase in [Ca2+]in. Analysis of Indo-1 fluorescence signals from neutrophils exposed to IIC, corrected for the contribution of the nonresponding population, indicated that every dose of IIC elicited a similar maximum [Ca+2]in within the responding population. In contrast, the magnitude of the increase in [Ca2+]in elicited by low doses of SIC did become dependent on dose. Cells treated with pertussis toxin and exposed to IIC exhibited a normal [Ca2+]in response both in magnitude and expression. Therefore, [Ca2+]in responses induced by immune complexes are expressed by subpopulations of PMN, in a response which is dependent on the valency of the stimulus. In addition, pertussis toxin sensitive G protein(s) appear not to have a major role in IIC-induced [Ca2+]in changes, membrane potential changes, production of superoxide anions, and elastase release.     

Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein

Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.     

Flow cytometric kinetic measurements of neutrophil phospholipase A activation.

The interrelationships between activation of phospholipases and neutrophil stimulus-induced Ca2+ responses remain unclear. We report here that immune complexes activate a phosphatidylcholine-specific phospholipase A in a neutrophil only after the cytoplasmic Ca2+ transient has been initiated in the same cell, while chemotactic peptide activation does not proceed via such a phospholipase A-mediated mechanism. Measurements of [Ca2+] changes and of phosphatidylcholine-specific phospholipase A activity were made by flow cytometry, using Indo-1 for Ca2+ indication, and a new fluorescent probe, bis-BODIPY-phosphatidylcholine, localized in the inner leaflet of the plasma membrane, to measure phospholipase A activation. Both 100 nM formyl-methionyl-leucyl-phenylalanine (with or without cytochalasin B) and 60 micrograms/ml insoluble immune complexes elicited cytoplasmic Ca2+ transients, but only insoluble immune complexes stimulated phospholipase A activation in a subpopulation of cells exhibiting an elevation of [Ca2+]in. Phospholipase A activation followed the Ca2+ transient, starting, in each cell, after [Ca2+]in had begun to decrease as Ca2+ redistributed in the activated cell. The products of this phospholipase activation were confirmed by thin layer chromatography. We conclude that neutrophils respond to immune complexes with an elevated cytoplasmic Ca(2+)-requiring phosphatidylcholine-specific phospholipase A activation and to chemotactic peptides by a different mechanism.     

Fluoride-mediated activation of the respiratory burst in electropermeabilized neutrophils

Electropermeabilization creates small pores in the plasma membrane allowing the introduction of low-molecular-weight modulatory components, such as ions and nucleotides, into the cytosol. The present study investigates fluoride-mediated stimulation of the signal transduction pathway that activates the respiratory burst in electropermeabilized neutrophils. In marked contrast to intact (i.e., non-electropermeabilized) neutrophils, cells permeabilized by this technique demonstrated an immediate and potent stimulation of the superoxide (O2-)-generating NADPH oxidase in response to the addition of fluoride. Furthermore, permeabilization of neutrophils in the presence of exogenously added ATP enhanced the rate of F(-)-mediated O2- production. Fluoride-stimulated O2- production in electropermeabilized neutrophils was antagonized by GDP beta S and dependent upon the presence of Mg2+ in the medium, but was insensitive to pertussis toxin treatment, consistent with the hypothesis that fluoride activates a G protein, probably Gp, by interacting with the nucleotide-binding site on the G alpha subunit. In addition, electropermeabilized neutrophil O2- release triggered by F- was blocked by staurosporine and H-7, indicating that this pathway proceeds largely through protein kinase C activation. However, nucleotide-enhanced O2- production was only partially blocked by these inhibitors, suggesting that under such conditions ATP either competes with the inhibitor-protein kinase interaction or affects the signaling pathway(s) in such a way that protein kinase C may no longer be necessary for the activation of NADPH oxidase.     

Differences in signal transduction between Fc gamma receptors (Fc gamma RII, Fc gamma RIII) and FMLP receptors in neutrophils. Effects of colchicine on pertussis toxin sensitivity and diacylglycerol formation.

Studies on the role of microtubule integrity in stimulus-response coupling in neutrophils have generated contradictory data. To determine the role of microtubule integrity in stimulus-response coupling elicited by two different mechanisms, i.e., engagement of the Fc receptors (FcR gamma II, FcR gamma III) or engagement of the receptor for FMLP, we utilized colchicine (10 microM), which reduces pericentriolar microtubules to 29% of control, and compared its effect with that of nocodazole (50 microM) and lumicolchicine (10 microM). We now demonstrate that treatment of neutrophils with colchicine but not lumicolchicine, inhibits degranulation elicited by engagement of Fc receptors but augments degranulation in response to FMLP. In contrast to the ligand-specific effect of microtubule-disruption on degranulation, superoxide anion production (assembly of the NADPH oxidase) is unaffected by colchicine regardless of the ligand. To determine whether intact microtubules were required for responses elicited by ligation of Fc gamma RII(CD32) or Fc gamma RIII(CD16), mAb directed against these receptors were employed. Treatment of neutrophils with mAb KuFc79 directed against Fc gamma RII(CD32) or mAb 3G8 directed against Fc gamma RIII(CD16) inhibited degranulation of neutrophils elicited by immune complexes (IC). In contrast, removal of most of Fc gamma RIII by phosphatidylinositol-specific phospholipase C did not significantly alter degranulation in response to IC. We conclude that degranulation elicited by IC results from ligation of both Fc gamma RII and phosphatidylinositol-specific phospholipase C-insensitive Fc gamma RIII. The importance of microtubule integrity on the generation of intracellular signals was also examined. Degranulation of neutrophils proceeds via pertussis toxin-sensitive and insensitive pathways; treatment of cells with colchicine did not augment the action of pertussis toxin. Stimulation of neutrophils by chemoattractants results in a biphasic increase in 1,2-sn-diacylglycerol; a rapid increase ("triggering") secondary to the action of a phosphatidylinositol-specific phospholipase C, and a late increase ("activation") secondary to the action of a phosphatidylcholine-specific phospholipase C. Treatment of cells with colchicine altered the production of both [3H]-arachidonic acid-diacylglycerol and diacyl[14C]glycerol in parallel to its effect on degranulation. These studies indicate that the requirement of intact microtubules for degranulation is ligand-specific. Furthermore, assembly of the respiratory burst oxidase does not require intact microtubules. Microtubules most likely alter the cycling of specific receptors or the generation of specific intracellular signals required for stimulus-response coupling in the course of degranulation. Intact microtubules are not uniformly required for the discharge of granule contents during exocytosis.     

Signal transduction by tumor necrosis factor alpha is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1.

Transmembrane signalling mechanisms of tumor necrosis factor alpha (TNF alpha) were examined with special reference to the involvement of G-protein, in intact and permeabilized murine osteoblast-like cells. TNF alpha stimulated the release of 3H radioactivity from intact cells labeled with [3H]arachidonic acid within 10 min in a dose dependent manner and the production of lyso forms of phospholipids, an event presumably mediated through the activation of phospholipase A2. Production of cAMP and inositol 1,4,5-trisphosphate was not affected by TNF alpha. Pretreatment of the cells with pertussis toxin inhibited the liberation of [3H]arachidonate. GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) reduced the binding affinity of [125I]TNF alpha to beta-escin-permeabilized cells. The addition of TNF alpha together with an unhydrolyzable analog of GTP, GTP gamma S, to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid led to a release of the 3H radioactivity. The production of prostaglandin E2 (PGE2) was markedly stimulated by TNF alpha in a dose over 100 ng/ml, with a latent time of about 3 h, and the stimulation was abolished by pretreatment with pertussis toxin. The time and dose requirements for this process differed from those for the possible activation of phospholipase A2, thereby indicating that other process(es) in addition to the activation of phospholipase A2 may be responsible for the enhanced production of PGE2. The activity of cyclooxygenase (i.e. the combined activities of prostaglandin endoperoxide syntase and PGH2-PGE2 isomerase) was stimulated by TNF alpha with much the same time and dose requirements as for the production of PGE2, and the activation was found to be due to the increased amount of the enzyme, as assessed by a Western blot analysis with anti-cyclooxygenase antibody. This process was also sensitive to pertussis toxin. Therefore, receptors for TNF alpha in MC3T3-E1 cells apparently couple to G-protein sensitive to pertussis toxin and the coupling regulates the activations of phospholipase A2 and the de novo synthesis of cyclooxygenase.     

Cholinergic-induced [3H] noradrenaline release in rat brain cortical slices is mediated via a pertussis toxin sensitive GTP binding protein and involves activation of protein kinase C

The involvement of a GTP-binding protein (G-protein) in the process of neurotransmitter release was examined using pertussis toxin and cholera toxin. Cholinergic agonists are shown to mediate [3H]noradrenaline release in rat brain slices via a pertussis toxin (1.2 micrograms/ml) sensitive, and cholera toxin (0.5 microgram/ml) insensitive G-protein. An indication for the involvement of a G-protein and phospholipase C activation in the release process was implied from the inhibitory effect of neomycin on K+-, veratridine- and carbachol-induced-norepinephrine release. Depolarizing agents mediate a neomycin-sensitive release, which is not which is not affected either by pertussis toxin or cholera toxin, suggesting a different mode of phospholipase C activation, unlike carbachol-induced release, which is both neomycin and pertussis toxin sensitive. Similarly, a hormone-sensitive carrier activated by phenylephrine not via alpha 1-adrenergic receptors, mediates a non-exocytosis efflux which is not affected by neomycin and is shown to be pertussis toxin-insensitive. The inhibitory action of protein kinase C inhibitors polymyxin B, K252a and H-7 [(1-(5-isoquinolinesulphonyl)-2-methyl-piperazine] on release, strongly suggests its participation in the process. Polymyxin B, a relatively selective protein kinase C inhibitor, inhibited carbachol-induced release (IC50 = 0.53 microM) as well as the K+ and the veratridine induced [3H] noradrenaline release, K252a, an inhibitor of various protein kinases at the ATP site, and H-7, another protein kinase C inhibitor, inhibited carbachol-induced noradrenaline released with IC50 = 35 nM and 3 microM respectively. Consistent with its inability to activate phospholipase C, phenylephrine-induced noradrenaline efflux was unaffected by polymyxin B (greater than 70 microM). These results offer more supportive evidence for a major role played by the dual messengers inositol trisphosphate and diacylglycerol (IP3/DG) in the mechanisms of neuronal release.     

Sphingosine-1-phosphate activates phospholipase D in human airway epithelial cells via a G protein-coupled receptor

Sphingosine-1-phosphate (SPP) acts as a first messenger in immortalized human airway epithelial cells (CFNPE9o(-)), possibly interacting with an Edg family receptor. Expression of the SPP receptors Edg-1 and Edg-3, as well as a low level of Edg-5/H218, was detected in these cells, in agreement with their ability to specifically bind SPP. The related lipids, lysophosphatidic acid and sphingosylphosphorylcholine, were unable to displace SPP from its high affinity binding sites, suggesting that the biological responses to these different lysolipids are mediated by distinct receptors. SPP markedly inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner and caused a remarkable elevation of intracellular calcium, both effects being sensitive to pertussis toxin treatment. Most importantly, SPP stimulated phosphatidic acid formation, which was maximal after 2 min and decreased within 8-10 min. In the presence of butan-1-ol, suppression of SPP-induced phosphatidic acid formation and production of phosphatidylbutanol were found, clearly indicating activation of phospholipase D (PLD). This finding was also confirmed by analysis of the fatty acid composition of phosphatidic acid, showing an increase in the monounsaturated oleic acid only. The decrease of phosphatidic acid level after 8-10 min incubation with SPP was accompanied by a parallel increase of diacylglycerol production, which was abolished in the presence of butan-1-ol. This result indicates that activation of phospholipase D is followed by stimulation of phosphatidate phosphohydrolase activity. Phosphatidic acid formation was insensitive to protein kinase C inhibitors and almost completely inhibited by pertussis toxin treatment, suggesting that SPP activates phospholipase D via a G(i/o) protein-coupled receptor.