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Cyclic AMP-increasing Agents Interfere with Chemoattractant-induced Respiratory Burst in Neutrophils as a Result of the Inhibition of Phosphatidylinositol 3-Kinase Rather than Receptor-operated Ca2+ Influx (∗)

  • Mosleh U. Ahmed
    Footnotes
    Affiliations
    Ui Laboratory, the Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi 351-01

    Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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  • Kaoru Hazeki
    Footnotes
    Affiliations
    Ui Laboratory, the Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi 351-01

    Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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  • Osamu Hazeki
    Affiliations
    Ui Laboratory, the Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi 351-01

    Ui Laboratory, the Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi 351-01
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  • Toshiaki Katada
    Affiliations
    Ui Laboratory, the Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi 351-01

    Ui Laboratory, the Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi 351-01
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  • Michio Ui
    Correspondence
    To whom correspondence should be addressed: the Ui Laboratory, the Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi 351-01, Japan. Tel.: 81-48-462-1111 (ext. 3431); Fax: 81-48-462-4693
    Affiliations
    Ui Laboratory, the Institute of Physical and Chemical Research, Hirosawa 2-1, Wako-shi 351-01

    Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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  • Author Footnotes
    § The first two authors contributed equally to this work.
    ∗ The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:October 06, 1995DOI:https://doi.org/10.1074/jbc.270.40.23816
      Superoxide anion and arachidonic acid were produced in guinea pig neutrophils in response to a chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP). Both responses were markedly, but the former response to a phorbol ester was not at all, inhibited when the cellular cAMP level was raised by prostaglandin E1 combined with a cAMP phosphodiesterase inhibitor. Increasing cAMP was also inhibitory to fMLP-induced activation of phosphatidylinositol (PI) 3-kinase and Ca2+ influx without any effect on the cation mobilization from intracellular stores. The fMLP-induced respiratory burst was abolished when PI 3-kinase was inhibited by wortmannin or LY294002, but was not affected when Ca2+ influx was inhibited. On the contrary, fMLP released arachidonic acid from the cells treated with the PI 3-kinase inhibitors as well as from nontreated cells, but it did not so when cellular Ca2+ uptake was prevented. The chemotactic peptide activated PI 3-kinase even in cells in which the receptor-mediated intracellular Ca2+ mobilization and respiratory burst were both abolished by exposure of the cells to a permeable Ca2+-chelating agent. Thus, stimulation of fMLP receptors gave rise to dual effects, activation of PI 3-kinase and intracellular Ca2+ mobilization; both effects were necessary for the fMLP-induced respiratory burst. Increasing cellular cAMP inhibited the respiratory burst and arachidonic acid release as a result of the inhibitions of PI 3-kinase and Ca2+ influx, respectively, in fMLP-treated neutrophils.

      INTRODUCTION

      The intracellular signaling pathways responsible for the formyl-methionyl-leucyl-phenylalanine (fMLP)
      The abbreviations used are: fMLP
      formyl-methionyl-leucyl-phenylalanine
      BAPTA
      O,O‘-bis(2-aminophenyl)ethylene glycol-N,N,N‘,N‘-tetraacetic acid
      Bt2cAMP
      dibutyryl cAMP
      [Ca2+]
      intracellular free Ca2+ concentration
      Fura2/AM
      Fura2 acetoxymethyl ester
      IBMX
      3-isobutyl-1-methylxanthine
      PGE1
      prostaglandin E1
      PI
      phosphatidylinositol
      PMA
      phorbol 12-myristate 13-acetate
      PIP3
      inositol 1,4,5-trisphosphate
      MAP
      mitogen-activated protein.
      -induced respiratory burst in phagocytes remain to be fully understood as yet, although the activation mechanism of the respiratory burst oxidase is currently elucidated as an assembly of a number of membrane-bound and cytosolic components, including gp91phox, p22phox, p47phox, p67phox, and Rac (see (
      • Chanock S.J.
      • El Benna J.
      • Smith R.M.
      • Babior B.M.
      ) for review).
      In general, one of the best strategies for identification of essential cellular signals involved in a cell response to a receptor stimulation is to search for the target with which an inhibitor interacts to abolish the response efficiently. Pertussis toxin was a good inhibitor and was successfully applied to the fMLP-induced respiratory burst. Prior exposure of guinea pig neutrophils to low concentrations of pertussis toxin for several hours prevented the cells from producing superoxide anion in response to the subsequent addition of fMLP(
      • Okajima F.
      • Katada T.
      • Ui M.
      ). The prevention resulted from the toxin-induced ADP-ribosylation of G protein (Gi-2), the chemical modification by which the modified G protein is uncoupled from receptors. Evidence has been thus provided for involvement of the G protein in the fMLP-induced respiratory burst via phospholipase C activation(
      • Okajima F.
      • Katada T.
      • Ui M.
      ,
      • Ohta H.
      • Okajima F.
      • Ui M.
      ,
      • Kikuchi A.
      • Kozawa O.
      • Kaibuchi K.
      • Katada T.
      • Ui M.
      • Takai Y.
      ).
      An additional example of useful inhibitors of the phagocytic respiratory burst is wortmannin, a fungal metabolite with hydrophobic sterol structure. Baggiolini and his colleagues (
      • Baggiolini M.
      • Dewald B.
      • Schnyder J.
      • Ruch W.
      • Cooper P.H.
      • Payne T.G.
      ,
      • Dewald B.
      • Thelen M.
      • Baggiolini M.
      ) first reported that incubation of human neutrophils with wortmannin or 17-hydroxywortmannin for 5 min inhibited fMLP-induced O2- generation and degranulation without affecting the chemoattractant-induced Ca2+ mobilization. The inhibition induced by wortmannin was so selective that it did not inhibit similar responses of neutrophils to a phorbol ester, a direct inhibitor of protein kinase C. Wortmannin has lately been proven to abolish the cellular signaling as a result of its direct interaction with phosphatidylinositol (PI) 3-kinase, providing convincing evidence for essential roles of this enzyme or the products of the enzyme in signaling pathways connecting G protein-coupled receptors and the respiratory burst(
      • Arcaro A.
      • Wymann M.P.
      ,
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ).
      The purpose of the present paper is further application of this useful strategy to identification of important signaling pathways responsible for the fMLP-induced respiratory burst in guinea pig neutrophils. Increases in cAMP within cells have long been known to inhibit receptor-mediated respiratory bursts in reticulocytes and macrophages (e.g.(
      • Takenawa T.
      • Ishitoya J.
      • Nagai Y.
      ) and (
      • DeTogni P.
      • Cabrini G.
      • DeVirgilio F.
      ) and references cited therein). We have identified intracellular signals that were impaired in parallel with the inhibition of fMLP-induced O2- generation upon the addition of cAMP-increasing agents to the cells.

      EXPERIMENTAL PROCEDURES

      Materials

      Materials were obtained from the following sources: cAMP, Bt2cAMP, fMLP, IBMX, PGE1, PMA, cytochrome c, A23187, staurosporine, and bovine serum albumin from Sigma; Fura2/AM and BAPTA/AM from Dojin Laboratories (Kumamoto, Japan); bisindolylmaleimide (GF109203X) from Calbiochem; 32Pi and [3H]arachidonic acid from DuPont NEN. Wortmannin and pertussis toxin were kind gifts from Drs. Y. Matsuda (Kyowa Hakko Kogyo Co.) and M. Tamura (Kaken Seiyaku Co.), respectively. The stock solution (10 mM) of wortmannin was prepared in dimethyl sulfoxide, stored at −20°C, and diluted with appropriate buffer immediately before use.

      Cells

      Neutrophils were isolated from peritoneal exudates of Hartley guinea pigs of 400 g body weight 18 h after intraperitoneal injection of sodium casein, and cultured for 4 h with or without 50 ng/ml of pertussis toxin at 37°C in the RPMI 1640 medium under 95% air, 5% CO2, as described previously(
      • Okajima F.
      • Ui M.
      ). The cells were loaded with [3H]arachidonic acid (carrier-free, 1 μCi/5 × 107 cells/dish) or Fura2/AM (3 μM) for 4 h or for a final 1 h, respectively, during the culture for the purpose of monitoring arachidonic acid release and [Ca2+]i changes subsequently. Following the culture, the monolayer was washed with the Ca2+-free Hepes-buffered medium which had been prepared by replacing CaCl2 by 1 mM EGTA in the regular Krebs-Ringer-Hepes medium (134 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 5 mM glucose, 0.2% bovine serum albumin, 20 mM Hepes, pH 7.4). The cells were then scraped off with a rubber policeman, washed, and finally suspended in the regular Krebs-Ringer-Hepes medium to be subjected to further analyses. Freshly isolated (noncultured) cell suspensions were used in some experiments in which cells were not intended to be loaded with the toxin, the fluorescent dye, or the radioactive metabolite; essentially the same results were obtained between the fresh and cultured cells as to O2- generation, 32P labeling, and their susceptibility to stimulants or inhibitors studied.

      Superoxide Generation

      The suspension of neutrophils (106 cells/0.5 ml) was first incubated for 10 or 15 min at 37°C in the presence or absence of inhibitors or cAMP-generating agents (henceforth both referred to solely as inhibitors for brevity) in the regular Krebs-Ringer-Hepes medium before being then stimulated with fMLP or PMA in the presence of 80 μM cytochrome c. The stimulation was stopped by adding ice-cold Ca2+-free Krebs-Ringer-Hepes medium (fortified with 10 mM EGTA), and the mixture was quickly centrifuged (1,500 × g for 10 s) to afford the supernatant to be assayed for O2- generation by measurement of the reduction of cytochrome c based on the difference spectrum at 550-540 nm.

      Measurement of [Ca2+]i

      The Fura2-loaded neutrophils (5 × 106 cells/0.5 ml), after being exposed to vehicles or inhibitors for 10 or 15 min at 37°C, were analyzed, with the additions shown in the figures, for Fura2 fluorescence at 510 nm with the excitation at 340 and 380 nm in the regular or Ca2+-free Krebs-Ringer-Hepes medium. Fluorescence was determined in a dual excitation wavelength fluorometer (Hitachi, F-2000) and expressed as a ratio of the fluorescence at the two excitation wavelengths to calculate [Ca2+] according to the equation described previously(
      • Grynkiewicz G.
      • Poenie M.
      • Tsien R.Y.
      ).

      Arachidonic Acid Release

      Aliquots (4 × 106 cells/0.5 ml) of [3H]arachidonate-labeled neutrophil suspensions, prepared as described above, were first exposed to inhibitors for 10 min and then incubated for 15 min with stimulants at 37°C. The incubation was stopped by the addition of 0.5 ml of Ca2+-free Krebs-Ringer-Hepes medium fortified with 10 mM EGTA. This was followed by immediate centrifugation to give the supernatant, the radioactivity in which, was determined as arachidonate release from the cells.

      32P Labeling of Phospholipids as a Measure of PI 3-Kinase in Intact Cells

      Production of 32P-labeled PIP3 was estimated by the method described previously(
      • Traynor-Kaplan A.E.
      • Thompson B.L.
      • Harris A.L.
      • Taylor P.
      • Omann G.M.
      • Sklar L.A.
      ). Neutrophil suspensions (108 cells/ml) were labeled with 32Pi (150 μCi/ml) by 30-min incubation at 30°C in the medium consisting of 10 mM Hepes-NaOH, pH 7.4, 136 mM NaCl, 4.9 mM KCl, and 5.5 mM glucose. These labeled cells were washed twice, resuspended at the density of 107 cells/ml in the regular Krebs-Ringer-Hepes medium, and, as 250-μl aliquots, incubated at 37°C for 10 or 15 min with or without inhibitors before the final stimulation with 0.1 μM fMLP for 15 s or longer. After stimulation, cells were lysed by vigorous stirring in 1.55 ml of chloroform/methanol/8% HClO4 (50:100:5), followed by addition with 1 ml of the chloroform/8% HClO4 (1:1) mixture to separate the organic phase, which was washed with chloroform-saturated 1% HClO4 and dried in vacuo. The lipids were dissolved in 20 μl of chloroform/methanol (95:5) and spotted on a thin-layer plate (Silica Gel 60, Merck), which had been impregnated with potassium oxalate by the procedure of development in a solvent system of 1.2% potassium oxalate-containing methanol/water (2:3) and activated by heating at 110°C for 20 min before spotting. The plate was developed in chloroform/acetone/methanol/acetic acid/water (80:30:26:24:14), dried, and visualized for the radioactivities with a Fuji BAS2000 Bioimaging Analyzer.

      Measurement of cAMP

      Incubation of neutrophil suspensions with 0.3 mM IBMX was stopped by the addition of HCl to make the final concentration of 0.1 N, which was followed by maintaining the dishes in boiling water for 3 min and centrifugation. The resultant supernatant was submitted for radioimmunoassay of cAMP as described previously(
      • Okajima F.
      • Ui M.
      ).

      RESULTS

      Selective Inhibition of Respiratory Bursts by cAMP-increasing Agents and Various Inhibitors

      Fig. 1shows typical O2--generating responses of guinea pig neutrophils to additions of increasing concentrations of fMLP and PMA. The concentration-dependent increases in O2- generation were observed for fMLP and PMA with the maximal responses being at 100 and 10 nM, respectively. The cell responses to fMLP were markedly reduced (Fig. 1A), but those to PMA were not affected (Fig. 1B), when the cells had been exposed for 10 min to agents that can increase the cellular content of cAMP (an adenylate cyclase activator, PGE1, an inhibitor of cAMP-hydrolyzing phosphodiesterase, IBMX, and their combination) or a cell-permeable cAMP analogue (Bt2cAMP). Actually, the cAMP content in neutrophils was less than 1, 6.2 ± 0.1, and 91 ± 4.2 pmol/107 cells (with the number of observations of six) after 10-min incubation without any addition, with 0.3 mM IBMX alone and its combination with 10 μM PGE1, respectively, under these conditions. Prior exposure of neutrophils to pertussis toxin also abolished the cell response to fMLP without affecting the response to PMA (data not shown).
      Figure thumbnail gr1
      Figure 1:Inhibition of fMLP-induced O2- generation by cAMP-generating agents. Freshly prepared (noncultured) guinea pig neutrophils were incubated with cAMP-increasing agents such as 10 μM PGE1 (▵), 1 mM Bt2cAMP (▪), 0.3 mM IBMX (•), 10 μM PGE1plus 0.3 mM IBMX (▴) or vehicle alone (○) for 10 min, before the subsequent 15-min incubation with increasing concentrations of fMLP (A) or PMA (B) and were then submitted for analyses of O2- generation as described under “Experimental Procedures.” Essentially the same results were obtained in three similarly designed experiments.
      The respiratory burst induced by fMLP was likewise inhibited by wortmannin (Fig. 2A) and staurosporine (Fig. 2B) in concentration-dependent manners. LY294002, a wortmannin-like inhibitor of PI 3-kinase(
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ), gave rise to effects, just like wortmannin(
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ), very similar to those caused by cAMP-increasing agents; fMLP-induced O2- generation was, but the PMA-induced generation was not at all, inhibited by the inhibitor (Fig. 2C). It is conceivable that the increase in cAMP may interact with the step that functions, like PI 3-kinase, upstream of protein kinase C in G protein-initiated signaling pathways. Bisindolylmaleimide, an inhibitor of protein kinase C much more specific than staurosporine(
      • Toullec D.
      • Pianetti P.
      • Coste H.
      • Bellevergue P.
      • Grand-Perret T.
      • Ajakane M.
      • Baudet V.
      • Boissin P.
      • Boursier E.
      • Loriolle F.
      • Duhamel L.
      • Charon D.
      • Kirilovsky J.
      ), inhibited, in concentration-dependent manners, the fMLP-induced respiratory burst as well as PMA-induced one (Fig. 2D). Thus, activation of protein kinase C, which was not susceptible to cAMP increases within cells or PI 3-kinase inhibitors such as wortmannin and LY294002, was essential for the G protein-initiated respiratory burst. In fact, staurosporine did, but wortmannin did not, inhibit PMA-induced O2- generation at all the concentrations of the phorbol ester employed (Fig. 2E).
      Figure thumbnail gr2
      Figure 2:Effect of inhibitors on fMLP-induced O2- generation in neutrophils. Fresh guinea pig neutrophils were first exposed for 10 min to increasing concentrations of wortmannin (A), staurosporine (B), LY294002 (C), or bisindolylmaleimide (D) before further 15-min incubation with 0.1 μM fMLP (•), 0.1 μM PMA (▴) or without addition (○). In E, the first exposure was done to 0.1 μM wortmannin (•), 0.1 μM staurosporine (▴) or vehicle (○), and the further incubation was with increasing concentrations of PMA. These cells were then assayed for O2- generation as described under “Experimental Procedures.” The same results were reproduced in additional two or three experiments.

      Inhibition by cAMP-increasing Agents of Receptor-operated Ca2+Influx

      Fura2-loaded neutrophils exhibited marked increases in [Ca2+]i in response to the addition of fMLP (Fig. 3). The increase in [Ca2+]i in response to fMLP was transient. The rapid increase was followed by a rapid decrease to form a sharp peak of [Ca2+]i within 30-60 s, after which [Ca2+]i leveled off at a much higher level than the prestimulation value in Ca2+-containing medium (Fig. 3A). Such sustained increases in [Ca2+]i were mostly due to continuous Ca2+ influx following receptor stimulation; the rapid [Ca2+]i peak became much smaller in magnitude in the incubation medium from which Ca2+ was nominally omitted (Fig. 3B). Addition of Ca2+, within 3 min after receptor stimulation, into the Ca2+-free medium caused a marked increase in [Ca2+]i (Fig. 3B). The Ca2+ addition to the cell suspension in the Ca2+-free medium, without receptor stimulation, did not cause any significant increase in [Ca2+]i (data not shown, but see a dotted trace in Fig. 4for example). Thus, the first and the second peaks of [Ca2+]i in Fig. 3B reflect mobilization of Ca2+ from the intracellular stores and Ca2+ influx from the exterior, respectively, in response of neutrophils to fMLP receptor stimulation.
      Figure thumbnail gr3
      Figure 3:Effects of cAMP-increasing agents and inhibitors on fMLP-induced [Ca2+]i increases. Guinea pig neutrophils were cultured for 4 h to be loaded with Fura2 as described under “Experimental Procedures.” In C and D, the culture medium was supplemented with 100 ng/ml of pertussis toxin. The loaded cells were incubated with 10 μM PGE1plus 0.3 mM IBMX (E and F), 1 μM wortmannin (G and H), 1 μM staurosporine (I and J), or vehicle (A, B, C, and D) for 10 min before being submitted to fluorescence analysis. The incubation medium used was the regular (Ca2+-containing) Krebs-Ringer-Hepes medium (A, C, E, G, and I) or the nominally Ca2+-free medium (B, D, F, H, and J). The addition of 0.1 μM fMLP was shown by the first arrowhead in each panel. In B, D, F, H, and J, CaCl2 was added as shown by the second arrowhead to make the final concentration of 2.5 mM. These tracings are representative of three experiments with similar results.
      Figure thumbnail gr4
      Figure 4:Failure of the cAMP-increasing agent to inhibit thapsigargin-induced Ca2+ influx. Fura2-loaded neutrophils, after first 10-min exposure to vehicle (A) or 10 μM PGE1plus 0.3 mM IBMX (B), were submitted to Ca2+ fluorescence analysis in the Ca2+-free medium as described for . In each panel, the first and the second arrowheads show the addition of thapsigargin (10 μM) and Ca2+ (2.5 mM), respectively. Dotted lines show [Ca2+]i observed without addition of thapsigargin (with the later Ca2+ addition only). Similar results were obtained in three separate experiments.
      When fMLP receptors were uncoupled from G proteins by prior exposure of cells to pertussis toxin, the agonist failed to increase [Ca2+]i significantly in either the presence (Fig. 3C) or the absence (Fig. 3D) of extracellular Ca2+. No significant [Ca2+]i increase occurred upon further addition of Ca2+ into the Ca2+-free medium bathing pertussis toxin- and fMLP-treated neutrophils, providing evidence for involvement of the toxin-sensitive G protein in the Ca2+ influx (Fig. 3D). In the cells pre-exposed to the cAMP-increasing agents (PGE1plus IBMX), the peak value of fMLP-induced [Ca2+]i increase was slightly smaller than the non-exposed cells and was followed by the return to the level much lower than that observed for control (Fig. 3E as compared with 3A), suggesting that receptor-coupled Ca2+ influx was inhibited upon increases in the cellular cAMP concentration. In fact, addition of fMLP into Ca2+-free medium caused the same increase in [Ca2+]i in the cells treated with cAMP-increasing agents as in control cells, whereas the subsequent addition of Ca2+ elicited much smaller increase in [Ca2+]i in the former cells than in the latter cells (Fig. 3F). Thus, elevation of intracellular cAMP attenuated fMLP receptor-operated Ca2+ influx without affecting the receptor-mediated mobilization of Ca2+ from the internal stores. There was only 20-30% decrease in fMLP-induced generation of inositol 1,4,5-trisphosphate, when guinea pig neutrophils had been exposed to the cAMP-generating agents (data not shown). Conceivably, such slightly attenuated generation of inositol 1,4,5-trisphosphate is still enough for the maximal mobilization of Ca2+ from the stores.
      The inhibition of receptor-operated Ca2+ influx was very unique to the action of cAMP-generating agents, in the sense that other inhibitors of fMLP-induced superoxide anion release, such as wortmannin and staurosporine, did not inhibit but rather enhanced the receptor-coupled Ca2+ influx (Fig. 3, G-J). Staurosporine may enhance Ca2+ entry by antagonizing protein kinase C-induced phosphorylation of proteins that is possibly involved in inhibition of capacitative Ca2+ influx in this cell type(
      • Montero M.
      • Garcia-Sancho J.
      • Alvarez J.
      ), although the mechanism for wortmannin-induced enhancement (Fig. 3H) remains to be clarified.
      The addition of Ca2+ following thapsigargin into neutrophil suspensions in the Ca2+-free medium gave rise to a marked increase in [Ca2+]i, despite lack of the increase without preaddition of the endoplasmic Ca2+ pump inhibitor, due to the capacitated Ca2+ entry following emptying of the intracellular stores but not mediated by receptor stimulation (Fig. 4). This capacitated Ca2+ entry was not affected significantly, or inhibited only slightly, by exposure of cells to cAMP-generating agents (Fig. 4). Emptying the Ca2+ stores after the thapsigargin treatment was evidenced by the failure of the treated cells to respond to fMLP by increasing [Ca2+]i (data not shown). Thus, the major target of increased cAMP or cAMP-dependent protein kinase appears to be the protein(s) involved in receptor-operated, rather than store-operated, Ca2+ channels.

      Ca2+Influx as a Mediator of Arachidonic Acid Release Rather than O2- Generation

      The chemoattractant-induced superoxide anion release was associated with an increase in Ca2+ influx (Fig. 3). Both effects of fMLP were markedly attenuated upon increases in intracellular cAMP. The increase in fMLP receptor-coupled entry of extracellular Ca2+, however, never played any essential role in O2- generation, which occurred and was inhibited by cAMP-increasing agents in the same magnitude in either the presence or absence of Ca2+ in the incubation medium (Fig. 5). In sharp contrast, arachidonic acid release provoked by fMLP receptor stimulation was highly dependent on the translocation of Ca2+ from the extracellular fluids (Fig. 6). No significant release of arachidonate was observed if the medium was depleted of Ca2+ (Fig. 6B).
      Figure thumbnail gr5
      Figure 5:fMLP-induced O2- generation and its susceptibility to cAMP-increasing agents in the presence or absence of extracellular Ca2+. Guinea pig neutrophils were first exposed for 10 min to 0.3 mM IBMX, 10 μM PGE1plus 0.3 mM IBMX, 1 mM Bt2cAMP or vehicle (none), as described in the top panel, and then incubated for 15 min with 0.1 μM fMLP, before being submitted to O2- assay as described under “Experimental Procedures.” The incubations were performed in the regular (Ca2+-containing) Krebs-Ringer-Hepes medium (A) or in the Ca2+-omitted medium (B). Similar results were obtained in two separate experiments.
      Figure thumbnail gr6
      Figure 6:Arachidonic acid release from neutrophils in the presence of cAMP-increasing agents or inhibitors. The [3H]arachidonate-labeled cells were first exposed for 10 min, as described in the left-hand panel, to 0.1 μM wortmannin, 10 μM PGE1plus 0.3 mM IBMX, 5 mM NiCl2, 5 mM CoCl2, 0.1 μM staurosporine, or vehicle (control) and further incubated for 15 min with 0.1 μM fMLP, 10 μM A23187, or vehicle (None), as shown at the bottom of columns, before being finally submitted to the arachidonic acid release assay according to the procedure described under “Experimental Procedures.” The incubations were conducted in the regular Krebs-Ringer-Hepes medium (A) or in the Ca2+-omitted medium (B). The data are representative ones from experiments performed more than three times with similar results.
      Again, wortmannin and cAMP-increasing agents exerted different effects on the response of neutrophils. The fungal metabolite did not inhibit arachidonic acid release under any condition tested, whereas PGE1plus IBMX was an inhibitor of the fMLP-induced release as strong as Ni2+ or Co2+, antagonists of transmembrane Ca2+ inflow (Fig. 6A). Ni2+ has been reported to be an inhibitor of receptor-operated Ca2+ entry(
      • Lynch J.W.
      • Lemos V.S.
      • Bucher B.
      • Stoclet J.C.
      • Takeda K.
      ). Staurosporine, like wortmannin, did not inhibit fMLP-induced arachidonic acid release. Failure of cAMP-increasing agents to inhibit Ca2+ ionophore-induced arachidonate production (Fig. 6A) is in agreement with the view that elevation of intracellular cAMP inhibits the fatty acid production as a result of the inhibition of receptor-coupled Ca2+ entry.

      Prevention of fMLP-induced PI 3-Kinase Activation in Cells Whose cAMP Content Was Raised

      32P-Loaded neutrophils were incubated with fMLP for a short time of 15 s to detect fMLP-induced PIP3 production as a result of PI 3-kinase activation (Fig. 7A). Progressively less activation of PI 3-kinase was evoked by fMLP as the concentration of PGE1 was increased from 0.01 to 10 μM in the IBMX-containing medium bathing the cells for 10 min prior to the addition of the chemoattractant. The range of the effective concentrations of PGE1 was essentially the same when comparison was made between PI 3-kinase activation (Fig. 7A) and O2- generation (Fig. 7B) as induced by fMLP. Moreover, the progressive inhibition of fMLP-induced O2- generation depending on increasing concentrations of PGE1 was inversely correlated with progressive increases of intracellular cAMP under the same conditions (Fig. 7B). Thus, the increase in intracellular cAMP antagonized both actions of fMLP to induce superoxide generation and to activate PI 3-kinase.
      Figure thumbnail gr7
      Figure 7:fMLP-induced PIP3 production and O2- generation in neutrophils and its progressive inhibition by increasing concentrations of PGE1 in the presence of IBMX. A, 32P-labeled neutrophils were first incubated with increasing concentrations of PGE1 for 10 min in the presence (+) or absence(-) of 0.3 mM IBMX and then incubated for 15 s with 0.1 μM fMLP or vehicle (None), as shown at the bottom of the panel, before being submitted to thin-layer chromatographic separation of phospholipids as described under “Experimental Procedures.” The incubations were done in the regular Krebs-Ringer-Hepes medium and the autoradiogram of the thin-layer plate is shown, in which the position of spots corresponding to PIP3 is indicated on the left. B, fresh neutrophils were first incubated with increasing concentrations of PGE1 in the presence of 0.3 mM IBMX and then stimulated by 0.1 μM fMLP (•) or not (○) under the same conditions as used for A, except for the stimulation time of 15 min instead of 15 s. Superoxide anion production was determined, as described under “Experimental Procedures,” and plotted as a function of PGE1 concentrations. The cellular content of cAMP after the 15-min incubation is also shown (▵). The results were reproduced in two or three separate experiments.

      PI 3-Kinase Activation and Intracellular Ca2+Mobilization Are Differentially Responsible for Superoxide Anion Generation

      No increase in [Ca2+]i was observed, even in the presence of the cation in the incubation medium, following fMLP addition to neutrophils that had been exposed to BAPTA/AM, a cell-permeable Ca2+-chelating agent (Fig. 8, A and B). The intracellular Ca2+ chelation abolished the superoxide-generating response of the cells to fMLP as well (Fig. 8D), but did not impair the chemoattractant-induced activation of PI 3-kinase (Fig. 8C). Such a manner in which BAPTA/AM inhibited fMLP-induced O2- generation formed a sharp contrast with the manner for the inhibition by cAMP-generating agents (Fig. 8D), which interfered with fMLP-induced PI 3-kinase activation under similar conditions (Fig. 8C) without affecting intracellular Ca2+ mobilization (Fig. 3F). Thus, the mobilization of Ca2+ from internal stores and the PI 3-kinase activation occurred in two separate signaling pathways emanating from fMLP receptor stimulation, eventually contributing to the same response of superoxide anion release from neutrophils. No arachidonate was released in response to fMLP from BAPTA-treated cells (data not shown), presumably because extracellular Ca2+ did not enter the cells under these conditions (Fig. 8B).
      Figure thumbnail gr8
      Figure 8:Differential effects of two respiratory burst inhibitors, an intracellular Ca2+-chelating agent and a cAMP-increasing agent, on fMLP-induced PI 3-kinase activation. Guinea pig neutrophils, Fura2-loaded (A and B), 32P-labeled (C), or nontreated (D), had been exposed for 15 min to 0.3 mM IBMX plus 10 μM PGE1 or 20 μM BAPTA/AM as described in these panels before further incubation with or without 0.1 μM fMLP in the Ca2+-containing regular medium. Tracings of [Ca2+]i, with the addition of fMLP as indicated by arrows, are shown in A and B. The incorporation of 32P into the PIP3 spots on the thin-layer plate during 15-s incubation with (hatched columns) or without (open columns) fMLP was determined as in and is illustrated in C. O2- generation during 15-min incubation with or without fMLP is likewise shown in D. Typical data are taken from experiments repeated twice with essentially the same results.

      DISCUSSION

      Different Signals Arising from fMLP Receptor Stimulation Are Responsible for Superoxide Generation and Arachidonate Release

      In the present paper, we measured two kinds of neutrophil responses, O2- generation and arachidonic acid release, to the addition of fMLP. Both responses were markedly attenuated by the agents (e.g. PGE1plus IBMX) causing accumulation of cAMP in cells. The other inhibitor of both responses was BAPTA/AM, a cell-permeable Ca2+-chelating agent, that abolished fMLP-induced intracellular Ca2+ mobilization and the subsequent Ca2+ influx (Fig. 8). It is also well known that prior exposure of neutrophils to pertussis toxin abolishes all the cell responses to fMLP including O2- generation and arachidonic acid release (see (
      • Ui M.
      ) for review). Other inhibitors (or maneuver of cells) tested here inhibited only either of these two responses. The chemoattractant-induced O2- generation was totally inhibited by protein kinase C inhibitors, staurosporine (and by bisindolylmaleimide also), and by direct and selective inhibitors of PI 3-kinase, wortmannin (and by LY294002 also), despite the failure of these inhibitors to affect fMLP-induced arachidonic acid release (Fig. 6). Vice versa, omission of Ca2+ from the incubation medium, just like the addition into the medium of Ni2+ or Co2+, direct inhibitors of transmembrane Ca2+ permeation, abolished arachidonate-releasing responses of neutrophils to fMLP and A23187, a Ca2+-ionophore (Fig. 6) without any modification of fMLP-induced O2- generation and its susceptibility to cAMP-increasing agent-induced inhibition (Fig. 5).
      It is very likely, therefore, that fMLP receptor stimulation triggered two signaling pathways each, with different susceptibility to certain inhibitors, separately leading to superoxide-generating and arachidonate-producing responses of neutrophils. fMLP is one of the chemokine families whose receptors possessing seven membrane-spanning regions release, upon being occupied by the ligand, the βγ-subunits from coupled pertussis toxin-sensitive G proteins. The signaling pathways may bifurcate at a point distal to the G protein, as evidenced by the fact that both responses are totally inhibited by treatment of the cells with pertussis toxin.

      Ca2+Influx as cAMP-susceptible Signals Essential for fMLP-induced Arachidonic Acid Release

      The Gβγ-protein is a direct activator of phospholipase Cβ(
      • Park D.
      • Jhon D.-Y.
      • Lee C.-W.
      • Lee K.-H.
      • Rhee S.G.
      ), a product of which, inositol 1,4,5-trisphosphate, causes mobilization of Ca2+ from the internal stores in the endoplasmic reticulum that is immediately followed by external Ca2+ influx across the plasma membrane through the receptor-operated and store-operated channels. The Ca2+ influx was essential for the observed arachidonate release, which was abolished by omitting Ca2+ from the medium or by inhibiting the flux by Co2+ or Ni2+ (Fig. 6). Stimulation of Ca2+ influx by a Ca2+-ionophore gave rise to arachidonic acid release. Cytosolic phospholipase A2 is likely to be activated as a result of Ca2+-dependent translocation into membranes where the substrate phospholipids are available(
      • Channon J.Y.
      • Leslie C.C.
      ,
      • Clark J.D.
      • Lin L.L.
      • Kriz R.W.
      • Ramesha C.S.
      • Sultzman L.A.
      • Lin A.Y.
      • Milona N.
      • Knopf J.L.
      ,
      • Lin L.L.
      • Wartmann M.
      • Lin A.Y.
      • Knopf J.L.
      • Seth A.
      • Davis R.J.
      ,
      • Nalefski E.A.
      • Sultzman L.A.
      • Martin D.M.
      • Kriz R.W.
      • Towler P.S.
      • Knopf J.L.
      • Clark J.D.
      ).
      Thus, it is very reasonable to assume that cAMP-increasing agents inhibit fMLP-induced arachidonic acid release as a result of their unique action to suppress Ca2+ influx following the mobilization of Ca2+ from internal stores (Fig. 3F), in accordance with previous reports(
      • Takenawa T.
      • Ishitoya J.
      • Nagai Y.
      ,
      • DeTogni P.
      • Cabrini G.
      • DeVirgilio F.
      ). It is tempting to speculate that receptor-operated Ca2+ channel is one of the targets of cAMP-dependent protein kinase, since cAMP-increasing agents were incapable of inhibiting thapsigargin-induced Ca2+ influx (Fig. 4) as well as Ca2+ ionophore-induced arachidonic acid release (Fig. 6A). Cyclic AMP has recently been reported to inhibit thrombin receptor-operated Ca2+ influx into platelets directly (
      • Doni M.G.
      • Cavallini L.
      • Alexandre A.
      ) or indirectly(
      • Heemskerk J.W.M.
      • Feijge M.A.H.
      • Sage S.O.
      • Walter U.
      ).

      Essential Role of cAMP-susceptible PI 3-Kinase in the fMLP-induced Respiratory Burst

      The respiratory burst was inhibited by both inhibitors of PI 3-kinase and protein kinase C when it was induced by fMLP, but was not inhibited by the lipid kinase inhibitors when it was evoked by PMA (Fig. 2). Likewise, cAMP-increasing agents, which inhibit fMLP-induced PI 3-kinase and O2- generation, exerted no effect on the PMA-induced respiratory burst (Fig. 1). Phosphorylation of cytosolic components of the respiratory burst oxidase, particularly p47phox, is one of the important activation mechanisms of the oxidase(
      • Chanock S.J.
      • El Benna J.
      • Smith R.M.
      • Babior B.M.
      ). At least 7 of the 11 serine residues on the p47phox molecule responsible for the activation were phosphorylated in PMA-activated neutrophils(
      • El Benna J.
      • Faust L.P.
      • Babior B.M.
      ), and the phosphorylation maintains the oxidase in the assembled/active state (
      • Curnutte J.T.
      • Erickson R.W.
      • Ding J.
      • Badwey J.A.
      ). Thus, PI 3-kinase, which is inhibited when cellular cAMP increased, is located upstream of protein kinase C in the signaling cascade arising from fMLP receptors and leading to the respiratory burst.

      Priming Role of Ca2+Mobilization for the fMLP-induced Respiratory Burst

      A number of recent reports have revealed that stimulation of fMLP receptors in neutrophils or neutrophil-type culture cell lines gives rise to phosphorylation of protein tyrosine residues(
      • Huang C.K.
      • Laramee G.R.
      • Casnellie J.E.
      ,
      • Grinstein S.
      • Furuya W.
      ,
      • Thompson H.L.
      • Shiroo M.
      • Saklatvala J.
      ), PI 3-kinase activation(
      • Arcaro A.
      • Wymann M.P.
      ,
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ,
      • Traynor-Kaplan A.E.
      • Thompson B.L.
      • Harris A.L.
      • Taylor P.
      • Omann G.M.
      • Sklar L.A.
      ,
      • Traynor-Kaplan A.E.
      • Harris A.L.
      • Thompson B.L.
      • Taylor P.
      • Sklar L.A.
      ,
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ,
      • Stephens L.
      • Jackson T.
      • Hawkins P.T.
      ,
      • Stephens L.R.
      • Jackson T.R.
      • Hawkins P.T.
      ,
      • Stephens L.
      • Eguinoa A.
      • Corey S.
      • Jackson T.
      • Hawkins P.T.
      ,
      • Fry M.J.
      ), and Ras activation leading to MAP kinase cascades via activation of Raf-1(
      • Grinstein S.
      • Furuya W.
      ,
      • Dusi S.
      • Donini M.
      • Rossi F.
      ,
      • Grinstein S.
      • Butler J.R.
      • Furuya W.
      • L'Allemain G.
      • Downey G.P.
      ,
      • Torres M.
      • Hall F.L.
      • O'Neill K.
      ,
      • Thompson H.L.
      • Marshall C.J.
      • Saklatvala J.
      ,
      • Worthen G.S.
      • Avdi N.
      • Buhl A.M.
      • Suzuki N.
      • Johnson G.L.
      ). It is unlikely that inositol 1,4,5-trisphosphate-induced Ca2+ mobilization functions downstream, or bifurcates from any site, of these tyrosine kinase-related signaling pathways, since IP3-generating phospholipase Cβ is directly activated by receptor-coupled Gβγ (
      • Park D.
      • Jhon D.-Y.
      • Lee C.-W.
      • Lee K.-H.
      • Rhee S.G.
      ). There are few papers reporting a decisive role of Ca2+ mobilization to trigger any tyrosine kinase signaling system. For instance, concanavalin A can activate a tyrosine kinase (
      • Asahi M.
      • Taniguchi T.
      • Hashimoto E.
      • Inazu T.
      • Maeda H.
      • Yamamura H.
      ) or MAP kinase (
      • Dusi S.
      • Donini M.
      • Rossi F.
      ,
      • Dusi S.
      • Rossi F.
      ) in Ca2+-depleted human neutrophils. Likewise, an increase in [Ca2+]i was not essential for fMLP-induced MAP kinase cascade activation(
      • Grinstein S.
      • Butler J.R.
      • Furuya W.
      • L'Allemain G.
      • Downey G.P.
      ). In fact, fMLP activated PI 3-kinase even in cells in which the chemoattractant did not cause Ca2+ mobilization, Ca2+ influx, or O2- generation after the treatment of cells with BAPTA/AM (Fig. 8C).
      Thus, a readily acceptable possibility is that internal Ca2+ mobilization primes, permits, or supports the signaling pathways responsible for O2- generation. In other words, the Ca2+ mobilization and the tyrosine kinase-related signalings including activation of PI 3-kinase are both indispensable for fMLP to provoke O2- generation. The small amount of Ca2+ mobilized in the Ca2+-free medium (e.g.Fig. 3B) would be enough to support the respiratory burst.

      The Site with Which an Increase in Cellular cAMP or cAMP-dependent Protein Kinase Interacts

      There is no evidence for direct phosphorylation by cAMP-dependent protein kinase (protein kinase A) of the p85 regulatory subunit or the p110 catalytic subunit of PI 3-kinase. Instead, a signaling pathway connecting the fMLP receptor stimulation and the PI 3-kinase activation appears to be a target of protein kinase A. Increasing cAMP or activation of cAMP-dependent protein kinase A has been reported to antagonize various receptor-initiated activation of MAP kinase cascades(
      • Worthen G.S.
      • Avdi N.
      • Buhl A.M.
      • Suzuki N.
      • Johnson G.L.
      ,
      • Graves L.M.
      • Bornfeldt K.E.
      • Raines E.W.
      • Potts B.C.
      • Macdonald S.G.
      • Ross R.
      • Krebs E.G.
      ,
      • Sevetson B.R.
      • Kong X.
      • Lawrence Jr., J.C.
      ,
      • Burgering B.M.T.
      • Pronk G.J.
      • Vanweeren P.C.
      • Chardin P.
      • Bos J.L.
      ,
      • Wu J.
      • Dent P.
      • Jelinek T.
      • Wolfman A.
      • Weber M.J.
      • Sturgill T.W.
      ,
      • Cook S.J.
      • McCormick F.
      ,
      • Hordijk P.L.
      • Verlaan I.
      • Jalink K.
      • Vancorven E.J.
      • Moolenaar W.H.
      ,
      • Vanrenterghem B.
      • Browning M.D.
      • Maller J.L.
      ,
      • Vaillancourt R.R.
      • Gardner A.M.
      • Johnson G.L.
      ,
      • Pyne N.J.
      • Moughal N.
      • Stevens P.A.
      • Tolan D.
      • Pyne S.
      ). The target of protein kinase A was located upstream of MAP kinase kinase(
      • Graves L.M.
      • Bornfeldt K.E.
      • Raines E.W.
      • Potts B.C.
      • Macdonald S.G.
      • Ross R.
      • Krebs E.G.
      ), between p21ras and Raf-1 (
      • Burgering B.M.T.
      • Pronk G.J.
      • Vanweeren P.C.
      • Chardin P.
      • Bos J.L.
      ,
      • Vanrenterghem B.
      • Browning M.D.
      • Maller J.L.
      ) or Raf-1 itself(
      • Worthen G.S.
      • Avdi N.
      • Buhl A.M.
      • Suzuki N.
      • Johnson G.L.
      ,
      • Wu J.
      • Dent P.
      • Jelinek T.
      • Wolfman A.
      • Weber M.J.
      • Sturgill T.W.
      ,
      • Cook S.J.
      • McCormick F.
      ,
      • Hordijk P.L.
      • Verlaan I.
      • Jalink K.
      • Vancorven E.J.
      • Moolenaar W.H.
      ,
      • Vaillancourt R.R.
      • Gardner A.M.
      • Johnson G.L.
      ). On the other hand, PI 3-kinase is directly activated by Gβγ in neutrophils (
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ) and platelets (
      • Thomason P.A.
      • James S.R.
      • Casey P.J.
      • Downes C.P.
      ) or by Ras(
      • Rodriguezviciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ), Rac(
      • Zheng Y.
      • Bagrodia S.
      • Cerione R.A.
      ), or Rho (
      • Zhang J.
      • King W.G.
      • Dillon S.
      • Hall A.
      • Feig L.
      • Rittenhouse S.E.
      ,
      • Kumagai N.
      • Morii N.
      • Fujisawa K.
      • Nemoto Y.
      • Narumiya S.
      ) proteins in various cell types. Alternatively, PI 3-kinase has been found to be located upstream of the Ras protein in certain growth factor-initiated signaling pathways(
      • Yamauchi K.
      • Holt K.
      • Pessin J.E.
      ,
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Varticovski L.
      • Harrison-Findik D.
      • Keeler M.L.
      • Susa M.
      ). Further studies are thus required before any decisive conclusion will be made as to how increasing cAMP inhibits fMLP-induced PI 3-kinase in neutrophils.

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