Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Possible existence of leukotriene D4 receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes P. N. Bouchelouche & D. Berild To cite this article: P. N. Bouchelouche & D. Berild (1991) Possible existence of leukotriene D4 receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes, Scandinavian Journal of Clinical and Laboratory Investigation, 51:sup204, 47-55, DOI: 10.3109/00365519109104594 To link to this article: http://dx.doi.org/10.3109/00365519109104594
Published online: 21 Mar 2011.
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Date: 15 March 2016, At: 12:30
Scand J Clin Lab Invest 1991; 51, Suppl. 204: 47-55
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
Possible existence of leukotriene D, receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes P. N. BOUCHELOUCHE' & D. BERILD*
' Fluorescence & Image Analysis Laboratory, Department of Clinical Chemistry, Department of Medical Gastroenterology C, Herlev Hospital, University of Copenhagen, Denmark Bouchelouche PN, Berild D. Possible existence of leukotriene D, receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes. Scand J Clin Lab Invest 1991; 51, Suppl. 204: 47-55. Previous work from this laboratory has demonstrated that stimulation of human polymorphonuclear leukocytes (PMN) with the peptidyl leukotriene D, (LTD,) results in the formation of second messenger signals, i.e. mobilization of intracellular free CaZt([Ca'+],)and hydrolysis of phosphoinositides (PIP,). Based on these earlier results we have employed radioligand binding techniques to study the presence of LTD, receptors in intact human PMN leukocytes. The binding of [3H]-LTD,to LTD, receptors is rapid, reversible, specific and saturable. Scatchard analysis of the binding data indicates the presence of 116-275 identical receptors per neutrophil with an apparent dissociation constant (KD)of 1,l-2,3 nM. Only one class of binding sites was identified. The LTD, receptors are located in the plasma membrane and are specific for LTD, since binding is unaffected by other leukotrienes. Furthermore, LTD, induces a rapid and persistent translocation of Protein Kinase C (PKC) from the cytosol to the membrane. The LTD, binding and PKC translocation could be blocked in a concentration dependent manner by the new and specific LTD, receptor antagonist ICI 198,615. These observations strongly suggest that human PMN might possess specific LTD, receptors which are coupled to the inositol trisphosphate pathway resulting in a rise in the cytoplasmic free Ca2+and redistribution of protein kinase C. A mechanism of signal transduction for leukotriene D, is proposed.
Key words: Leukotriene receptors; inflammation; intracellular free Ca2'; ICI 198,615; second messengers; inositol trisphosphate; protein kinase C. Reprints: Pierre N. Bouchelouche, M.D., Dept. of Clinical Chemistry, KAS Herlev, Herlev Ringvej 75, DK-2730 Herlev, Denmark.
48 P. N. Bouchelouche & D. Beriki
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
INTRODUCTION Arachidonic acid metabolites may generate a variety of hypersensitivity and inflammation reactions. The cysteinyl leukotrienes (LTC,, LTD,, and LTE,) represent one family of arachidonic acid metabolites with potent effects in many organs and tissues [ 11. Leukotriene D, (LTD,) is a potent bronchoconstrictor and has primarily been studied in regard to the human airway [2]. In addition to its spasmogenic effects, LTD, promotes mucus secretion, and the clinical interest in the cysteinyl leukotrienes in obstructive airway diseases is evidently great [2]. Some tissues have already been described regarding receptors for leukotriene D, including tissue from guinea-pig [3], human lungs [4], and rat basophilic leukemia cells [5], while their existence in human blood leukocytes has never been reported [6]. Recently, our team provided indirect evidence of the existence of specific LTD, receptors in human PMN in which activation led to rapid breakdown of inositol lipids and increase in the intracellular concentration of free Ca" [6]. Using radioligand binding assays for ['HILTD, and selective LTD, antagonists, we have studied the binding characteristics of [3H]-LTD, to humans PMNs. The results obtained correspond to the kinetic expected for membrane associated receptors probably using PI turnover as part of the signal transduction.
Groningen, The Netherlands). Purified human albumin (Behring Werke, Marburg, FRG). SR2640 (Leo Pharmaceutical, Ballerup, Denmark). Fura-2/acetoxymethylester (fura-2/AM) and the non-ionic detergent Pluronic F-127 were obtained from Molecular Probes (Junction City, OR, USA). Ethyieneglycol-bis-(b-aminoethy1ether)N,N,N,N-tetraacetic acid (EGTA), diethylenetriamine pentaacetic acid (DTPA), digitonin (stock solution 10 g/L), CaCl,, leupeptin, dithiothreitol, PMSF, trypsin inhibitor, benzamidine and triton X-100 were obtained from Sigma Chemical Company ( S t . Louis, MO, USA). The purity and concentration of EGTA was determined by the ammonium oxalate method [7] and by titration with Ca2' [8], respectively. [y-3zP]-ATPwas from D U PONT Denmark A/S. RPMI-1640 from Flow Laboratories, Scotland. ICI 198,615 was a gift from ICI Pharmaceuticals, Wilmington, Delaware, USA. Isolation of neutrophils Venous blood (EDTA 10 mM) obtained from the cubital vein of healthy volunteers who had taken no drugs for at least 2 weeks was sedimented on methylcellulose and buffy coat leucocytes were fractionated by gradient centrifugation on lymphoprep after Boyum [9]. Erythrocytes were removed by hypotonlysis. The final suspension contained > 97 % neutrophils with a viability > 95 % judged by the trypan blue extrusion method. The leukocytes were kept at 4 "C and used within one hour.
MATERIALS AND METHODS Reagents ['HI-LTD, at a specific radioactivity of 106 Ci/mmol to 225 Ci/mmol (Amersham International, Denmark). LTB,, LTC,, LTD,, LTE, (Paesel GmbH, Frankfurt am Main, FRG). Ethylenediaminetetraacetat(EDTA) (Merck), methylcellulose (Bie & Berntsen, Copenhagen, Denmark), Lymphoprep (Nygaard, Oslo, Norway). Dibutylphthalat and dinonylphthalat (Merck). Instagel (Packard,
Receptor studies A radioligand binding assay similar to that previously described for LTB, receptors [lo, 111was used. Duplicate samples of PMN in 200 pL Gey buffer were incubated with rH]-LTD, (0.1-2.5 nM) with or without a 1000-fold excess of LTD, to determine nonspecific binding. Samples of lo7 cells/mL were incubated for 60 minutes at 25 "C with intermittent shaking. Following incubation the suspension was rapidly centrifuged
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
Signal transduction of LTD, in human neutrophils.
through a precooled dibutyl/dinonylphthalat oil layer (3/1 v/v) for 30 s at 11000 g. The bottom of the tube with the PMN were cut off and radioactivity was determined in a Tracor Analytic Scintillation Counter with automatic quench correction. The existence of multiple binding sites was evaluated by using a fixed concentration of 2.5 nM [3H]LTD, and varying concentrations of unlabelled LTD, (0.1- 1 pM). The specificity of the binding sites was investigated using structural LTD, analogues (LTB,, LTC,, LTE,) and specific LTD, receptor antagonists. The existence of intracellular binding sites was elaborated in experiments on sonicated and saponin treated cells. Fluorometric measurements of intracellular free calcium Cytosolic free CaZ+was measured with the calcium sensitive fluorescent dye fura-2. Fura-2 ester was kept in stock solution (dimethylsulfoxide) and diluted in RPMI1640 medium immediately before use. In order to improve dye loading fura-2/AM was added briefly sonicated to obtain fine emulsions and an equal volume of the nonionic detergent Pluronic F-127 (25 % w/v) was added. Briefly described the cells (5.lo6cells/mL) were loaded by incubation for 30 min at 37 "C with 1 pM of the fura-2/AM mixture, washed twice to remove the extracellular dye and left at room temperature until use. The medium for fluorescence measurements consisted of (in mM): 145 NaCI, 5 KCl, 1 Na,HPO,, 1 CaCl,, 0.5 MgSO,, 5 glucose and 20 HEPES buffer. The medium was adjusted with NaOH to pH 7.4 at 37 "C. Time course of change in fluorescence was measured either by single (340 nm) or dual (340 and 380 nm) excitation wavelength spectrometry as previously described [ 12,131. The photometer was programmed to scan back and forth between the two excitation wavelengths and to reject data during the transition period. This technique is more cumbersome and was therefore only used in those experiments to determine the absolute
49
concentration of intracellular free Ca". The advantages of the dual-wavelength method have already been described [14]. Autofluorescence values were obtained from matching suspensions of unloaded cells from the same batch. The dissociation constant of the Caz'-fura-2 complex was assumed to be 224 nM [15]. None of the experimental agents used altered the autofluorescence of the cells. Protein kinase C translocation assay Buffer solutions: Buffer A contained (in mM): 20 TRIS-HCI, 10 EGTA, 4 EDTA and 157 mg/L benzamidine. pH was adjusted to 7.5. Buffer B contained (in mM): 20 TRIS-HCl, 5 EGTA, 2 EDTA and 157 mg/L benzamidine. Buffer C contained (in mM): 20 TRIS-HCL, 0.5 EGTA, 0.5 EDTA and 157 mg benzamidine. At the day of the experiment trypsin inhibitor (1 mg/mL), dithiothreitol (1 mM), PMSF (20 pL/mL) and leupeptin (3pLlmL) were added to all buffers. Intact human polymorphonuclear leukocytes (5.10' cells/mL) were preincubated in 3 mL RPMI-1640 with or without LTD, (lo9- lo' M). The reactions were stopped with 10 mL ice-cold RPMI-1640 medium at the time indicated in the figures. The cells were rapidly centrifugated at 300 g for 5 min. The cell pellets were resuspended in 2 mL buffer A and ultrasonically disrupted at 4 "C. The crude homogenates were centrifuged at 100.000 g for 60 min at 4 "C. After centrifugation, 2 mL buffer A containing 0.1 % triton X-100 was added to the supernatant (cytosol fraction). The pellet (membrane fraction) was resuspended in 4 mL buffer containing 0.1 % triton X-100, stored on ice for one hour and finally centrifuged at 100.000 g for 30 min. Membrane and cytosol fractions were chromatographed in parallel on DEAE-cellulose columns (1.4 cm) using an FPLC system (Pharmacia, Uppsala, Sweden). The columns were preequilibrated with 20 mL buffer A. There-
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
50 P. N. Bouchelouche & D. Berild after, the columns were washed with 10 mL buffer B, 10 mL buffer C and finally with 3 mL buffer C containing 25 mM NaC1. Protein kinase C and the phospholipidindependent PKC were eluted with 5 mL buffer C containing 100 and 400 mM NaCl, respectively. PKC activity was determinated by measurement of the transfer of "P from [y"P]ATF' to histone [16]. The reaction was stopped after 10 min at 30 "C with ice-cold ATP in excess and an aliquot of the mixture applied onto 3.3 cm' squares of Whatman P-81 chromatography paper. Following thorough washing in 50 mM NaCl and overnight drying radioactivity was determinated by scintillation counting. PKC activity was defined as the difference between the 32P incorporation in the presence and absence of phosphatidylserine and diolein [ 171.
FIG. 1. Association and dissociation curve for the specific binding of ['HI-LTD, to human PMN leucocytes. 2.5 nM [ HI-LTD, was added to lo7 PMN/mL and bound radioactivity was measured every 10 min. After 55 min excess of LTD, was added to the suspension and dissociation was measured. The curve is corrected for non-specific binding as assessed by addition of 2.5 pM unlabelled LTD,.
RESULTS
density (Bmax) of 116-275 binding sites per cell (n=3).
Optimal conditions for receptor binding studies were established at a cell concentration of lo7 PMN/mL and incubation at 25 "C for 60 min in a Gey buffer containing 0.1 % bovine albumin. Characterization of [)H]-LTD, binding to human PMN leukocytes The time course for binding of ['HI-LTD, to human PMNs is shown in Fig. 1. This figure also demonstrates the rapid reversibility caused by addition of excess unlabelled LTD,. Fig. 2A demonstrates a typical saturation curve for binding of [3H]-LTD,(0.1-2.0 nM) to PMN. Non-specific binding was linear with increasing concentrations of ['HI-LTD, (data not shown). In competition experiments using fixed concentrations of 2.5 nM ['HI-LTD, and varying concentrations of unlabelled LTD, (0.1 - 1 pM) no further binding sites were detected. Kinetic analysis using the Scatchard plot (Fig. 2B) revealed a dissociation constant (K,) of 1.1-2.3 nM with a receptor
-
1.01 .o
I
r
9 m 0 505
I
0
1
30
60
120 lio
Time (rnin)
Inhibition of [.'H]-LTD, binding to human PMNs by structurally related leukotrienes and LTD, receptor antagonists In competition experiments unlabelled LTD, inhibited [3H]-LTD,binding in a concentration dependent manner (Fig. 3). The structurally related leukotrienes LIB, and LTE4 did not influence LTD4binding, while LTC, inhibited at concentrations above 10" M. Fig. 4A shows that the ['HI-LTD, binding is inhibited in a concentration dependent manner by the specific LTD, receptor antagonist SR2640, while Fig. 4B demonstrates inhibition by the more potent LTD, receptor antagonist ICI 198,615 [18] resulting in approximately 90 % inhibition at a concentration of only 0.1 nM. LTD, receptor-mediated cytosolic free Ca" mobilization Human PMN (5.106cells/mL) loaded with fura-2 were suspended in calcium rich medium (left) and in calcium free medium (middle). LTD, was added as indicated. In Fig. 5 (right), human PMN was incubated
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
Signal transduction of LTD, in human neutrophils. 5 1
Concentration of LTDq (nM) FIG.2. A. Concentration dependence of the specific binding of ['HI-LTD, to human PMN leukocytes. lo7 PMN/mL were incubated with increasing concentrations of ['HI-LTD, at 25" for 60 min. The curve is corrected for nonspecific binding as assessed by addition of 2.5 gM unlabelled LTD,. B. Typical Scatchard plot derived from binding in Fig. 2.4.
(5 min) with the LTD, receptor-antagonist ICI 198,615 and then stimulated with LTD, as indicated. Typical trace of 10 experiments.
1.5
1.0.
r r L
Q 0.5
I
10
9
8
7
6
Concentration(.lqM)
FIG. 3 Competitive inhibition of ['HI-LTD, to human leukocytes by structurally related hg&ds. Suspensions of lo' PMN/mL were incubated at 25" for 60 min with 2 nM ['HI-LTD,. Non-radioactive LTB,, LTC,, LTD, and LTE, were added to the cell suspensions in concentration 10.'' M to lod M.
Protein kinase C activation. To investigate whether the stimulation of the LTD,-receptor was linked to the phospholipids-sensitiveCa*+-dependentprotein kinase C, we studied the translocation of this enzyme in the particulate and the cytosol fraction of human PMN treated with LTD,. PKC redistribution from cytosol to membrane is usually accepted as the first step of its activation [19]. As can be seen from Fig. 6.4, stimulation of the cells with LTD, provokes a rapid and marked reduction in the PKC activity in the cytosol and an increase in the membrane fraction. The translocation of PKC started immediately after addition of LTD, and reached a maximum within 5-10 min. Preincubation of the cells with 10 nM of the receptor antagonist ICI 198,615 com-
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
52 P. N. Bouchelouche & D.Berild
0
I
I
10
100
0
f
1Mxl
0
loo00
Concentration of SRZMO (nM)
01
02
03
04
Concentration of ICI 198,615 (nM)
FIG. 4. A. The influence of the specific LTD, receptor anta onist SR2640 on the binding of I3H]-LTD, to human PMN leucocytes. lo7 PMN/mL was incubated with 2.5 nM [5HI-LTD,. After equilibrium was achieved, increasing concentrations of the antagonist were added and the bound radioactivity measured. B. The influence of the more potent LTD, receptor antagonist ICI 198,615 on the percentage [’HI-LTD, bound to human PMN at equilibrium. The procedure was as in Fig. 4A.
[
); , : : ,
rm-
1’
-6M
I
t
10 nM
10 nM
10 nM
LTDI
LTDa
LTOi
FIG.5. LTD,-induced increase in PMN cytosolic free Ca”. Leff and middle: Intact human PMN loaded with fura-2 were suspended in medium containing 1 mM calcium and in calcium free medium, respectively. The cells were challenged with 10 nh4 LTD, and fluorescence was recorded. Right: Effect of the LTD, receptor antagonist ICI 198,615 on the maximum increase in [Ca”],. Each trace is representative of 3 separate experiments.
pletely abolished the translocation of the enzyme (Fig. 6B) DISCUSSION The possibility that human PMN leukocytes might express receptors for LTD, was initially suggested in our previous report on
LTD, induced increase in the level of intracellular free Ca2+,generation of inositol phosphates and down regulation of the human PMN migration response [6]. The finding that the LTD, receptor antagonist _ . . . ._. . - . . . SKZ640 is able to inhibit the Ll‘U,-induced generation of these second messengers and to suppress the attenuation by LTD, of LTB,-directed PMN chemotaxis [6], indicates that LTD,-receptors were involved. The presence of specific receptors for leukotriene D, in a number of tissues including human [4] and guinea pig lungs [3], human alveolar macrophages [20], and rat basophilic leukemia cells [5] has been demonstrated in a number of studies. Moreover, Bach et a1 have recently obtained preliminary evidence documenting the presence of specific LID, binding sites on cultured mesangial cells [21]. In the present study we used chemical and pharmacological methods to identify LTD, binding sites on human PMN, and it is shown for the first time that human PMNs might possess specific LTD, receptors at their plasma membrane. The binding is reversible and shows rapid saturation and a
Signal transduction of LTD, in human neutrophils. 53
Membrane
-0
lO?y
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
-6
-
cytosol
20
10
30
Time (min)
Time (min)
FIG. 6. A. Effect of LTD, on PKC redistribution from cytosol to membrane. PKC activity was determined in cytosolic and membrane fractions of human PMN treated with 0.1 pM LTD, at indicated times. Representative trace
of 2 experiments each assayed in triplicate. B. Effect of the LTD, receptor antagonist ICI 198,615 (10 nM)on PKC activity in LTD, stimulated human PMN. Representative trace of 3 experiments in triplicate.
FIG. 7. Model for signal transduction mechanism for
LTD, in human PMN leukocytes.
high affinity of ['HI-LTD, for the binding sites. The specificity of the LTD, receptors is supported by the lack of effect of LTB, and LTE, on ['HI-LTD, binding and the competitive inhibition by LTD, and by the LTD, receptor antagonists ICI 198,615 and SR2640. The enzymatic conversion of LTC, into LTD, may explain the small competitive inhibition of binding of ['HI-LTD, by LTC,. This could be due to the fact that PMN leukocytes are capable of both synthesis and degradation of LTC, [22]. Interaction of LTC, with y-glutamyltranspeptidase results in cleavage of glutamic acid and glycine to yield LTD, [23]. Permeabilization of the cells with the nonionic detergent saponin or disruption of the cells by sonication did not increase the number of binding sites, indicating that human PMN leukocytes possess no LTD,
receptors bound to intracellular organelles in contrast to LTC, [22]. Furthermore, the PMN leukocyte receptor for LTD, differs from those of LTB, with respect to affinity. The LTB, receptor consists of both high and low affinity receptors [ 101. Scatchard analysis of the specific binding of LTD, to the cells in this study revealed a single class of binding sites with very few receptors per cell compared to LTB, [lo]. Addition of LTD, to human PMN causes an immediate and transient increase in [Ca'+], in Ca rich as well as in Ca free media. The threshold concentration for effect was 10-l' M LTD, and maximal response was achieved at 10.' M LTD,. Furthermore, LTD, causes a translocation in PKC from the cytosol to the membrane in the concentration range 10"o-lO' M LTD,. The observation that the response in [Ca"], to LTD, can be obtained at concentrations very similar to the &-value for interaction between 'LTD, and its receptor, in addition to the inhibition of both receptor binding and response by the specific LTD, receptor antagonist ICI 198,615 strongly indicate that CaZt mobilization and PKC activation are mediated through binding of LTD, to the receptor. Previously, we have demonstrated that stimulation of human PMN with LTD4 resulted in the breakdown of phosphatidylinositoi bisphosphate. Therefore, it seems
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
54 P. N. Bouchelouche & D. Berild
likely that in human PMNs the binding of LTD, to its receptor results in the formation of IP,, which triggers the release of Ca2+ from intracellular stores, and translocation of protein kinase C. In conclusion, the simplest tentative model of the signal transduction system of the leukotriene D, receptor is illustrated in Fig. 7. LTD, binding to the receptor activates phospholipase C (PLC)at the plasma membrane. Activation of this enzyme leads to cleavage of phosphatidylbisphosphate(PIP,), resulting in the formation of two second messengers inositol 1,4,5-trisphosphate (IP,) and diacylglycerol (DG). Inositol 1,4,5trisphosphate mobilizes calcium from internal stores resulting in transient increase in the cytosolic free Ca2+,while diacylglycerol, ilicreases protein phosphorylation by activation of protein kinase C (PKC).
4.
5.
6.
I. 8.
9.
10.
ACKNOWLEDGEMENTS We wish to thank Mrs. Annie Petersen for her skilled assistance in the binding study experiments. We also thank ICI Pharmaceuticals Products, Wilmington, Delaware, USA for the kind gift of ICI 198,615 and Leo Pharmaceuticals Products, Denmark for the supply of SR2640. We further thank N.O. Christiansen and C.S. Larsen (Department of Medicine and Infectious Diseases, Marselisborg Hospital, k h u s C, Denmark) for familiarizing us with the field of protein kinase C.
11.
12.
13.
14.
15.
REFERENCES 1.
2. 3.
Samuelsson 8, Dahlen SE, Lmdgren JA, Rouzer CA, Serhan CN. Leukotrienes and Lipoxins: Structures, biosynthesis, and biological effects. Science 1987; 237 1171-6. Piper P. Formation and actions of leukotrienes. Physiol Rev 1984; 64: 744-61. Aharony D, Falcone RC, Krell D. Inhibition of 3H-leukotriene D, binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615. J Pharmacol Exp Ther 1987; 243: 921-6.
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Lewis MA, Mong S, Vessella RL, Crooke ST. Identification and characterization of leukotriene D, receptors in adult and fetal human lung. Biochem Pharmacol 1985; 34: 4311-7. Mong S, Wu HL, Wong A, Saran HM, Crooke ST. Leukotriene D4 receptor-mediated phosphoinositol hydrolysis and calcium mobilization in rat basophilic leukemic cells. J Pharmacol Exp Ther 1988, 247 803-13. Bouchelouche PN, Ahnfelt-RBnne I, Thomsen MK. LTD, increases cytosolic free calcium and inositol phosphates in human neutrophils: Inhibition by the novel LTD, receptor antagonist, SR2640, and possible relation to modulation of chemotaxis. Agents and Actions 1990; 29: 299307. Miller DJ, Smith GL. EGTA purity and the buffering of calcium ions in physiological solutions. Am J Physiol 1984; 246: C160-6. Moisescu DG, Push H. A pHmetric method for the determination of the relative concentration of calcium to EGTA (abstr.). Pfliigers Arch 1875; 355 R122. Boyum A. Separation of blood leucocytes, granulocytes and lymphocytes. Tissue Antigens 1973; 4 269-74. Goldman BW, Goetzl El. Heterogeneity of human polymorphonuclear leukocyte receptors for leukotriene B,. Identification of a subset of high affinity receptors that transduce the chemotactic response. J Exp Med 1984; 159 1027-41. Berild D, Bouchelouche PN, Skovgaard-Poulsen H, Elmgren J. Impaired cytosolic free Ca2+ response, chemotaxis and leukotriene B, receptor function in peripheral neutrophils from patients with Crohn's disease. Submitted for publication. Bouchelouche PN, Hainau B, Frederiksen 0. Effect of BAY K 8644 on cytosolic free calcium in isolated rabbit gall-bladder epithelial cells. Cell Calcium 1989; 10: 37-46. Bouchelouche PN, Reimert C, Bendtzen K. Effects of natural and recombinant interleukin la and -B on cytosolic free calcium in human and murine fibroblasts. Leukemia 1988,2 691-6. Tsien RY, Rink TJ, Poenie M. Measurement of of cytosolic free Ca'' in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 1985; 6: 145-53. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260:3440-50, Christiansen NO, Larsen CS, Juhl H, Esmann V. Membrane-associated protein b a s e s in phorbol ester-activated human polymorphonuclear leukocytes. Biochem Biophys Acta 1986; 884: 54-9. Su HD, Shoji M, Mazzei GJ, Vogler WR,Kuo JF. Effects of selenium compounds on phospholipids/Ca,+-dependent protein kinase C (protein kinase C) system from human leukemic cells. Cancer Research 1986; 46: 3684-7.
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Signal transduction of LTD, in human neutrophils. 55 18. Snyder DW, Gdes RE, Keith RA,Yee YK, Krell RD. The in vifro pharmacology of ICI 198,615: A novel, potent and selective peptide leukotriene antagonist. J Pharmacol Exp Ther 1987; 243 548-56. 19. Turner RS, Kuo JF. Phospholipids-sensitive Ca” -dependent protein b a s e C (protein kinase C): The enzyme, substrates, and regulation. In: Kuo JF (ed). Phospholipids and cellular regulation, vol 11. CRC Press, 1986. 20. Opmeer FA, Hoogsteden HC. Characterization of specific receptors for leukotriene D, on human alveolar macrophages. Prostaglandins 1984; 28. 183-95. 21. Bard KF, Ebert J, Hoover RL, Mong S, Harris RC. Characterization of leukotriene D, binding to rat glomerular mesanglial cells: demonstration of LTD,-stimulated inositol phosphate synthesis and ’H-thymidine incorporation. Kidney Int 1988, 3: 253 (abstr). 22. Baud L, Koo CH, Goetzl El. Specifity and cellular distribution of human polymorphonuclear leukocyte receptors for leukotriene C,. Immunology 1987; 6 2 53-9. 23. Krilis S, Lewis RA, Corey El, Austen FK. Bioconversion of C-6 sulfidopeptide leukotrienes by the responding guinea pig ileum determines the time course of its contraction. J Clh Invest 1983: 71: 909-15.
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Possible existence of leukotriene D4 receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes P. N. Bouchelouche & D. Berild To cite this article: P. N. Bouchelouche & D. Berild (1991) Possible existence of leukotriene D4 receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes, Scandinavian Journal of Clinical and Laboratory Investigation, 51:sup204, 47-55, DOI: 10.3109/00365519109104594 To link to this article: http://dx.doi.org/10.3109/00365519109104594
Published online: 21 Mar 2011.
Submit your article to this journal
Article views: 1
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [McMaster University]
Date: 15 March 2016, At: 12:30
Scand J Clin Lab Invest 1991; 51, Suppl. 204: 47-55
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
Possible existence of leukotriene D, receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes P. N. BOUCHELOUCHE' & D. BERILD*
' Fluorescence & Image Analysis Laboratory, Department of Clinical Chemistry, Department of Medical Gastroenterology C, Herlev Hospital, University of Copenhagen, Denmark Bouchelouche PN, Berild D. Possible existence of leukotriene D, receptors and mechanism of their signal transduction in human polymorphonuclear leukocytes. Scand J Clin Lab Invest 1991; 51, Suppl. 204: 47-55. Previous work from this laboratory has demonstrated that stimulation of human polymorphonuclear leukocytes (PMN) with the peptidyl leukotriene D, (LTD,) results in the formation of second messenger signals, i.e. mobilization of intracellular free CaZt([Ca'+],)and hydrolysis of phosphoinositides (PIP,). Based on these earlier results we have employed radioligand binding techniques to study the presence of LTD, receptors in intact human PMN leukocytes. The binding of [3H]-LTD,to LTD, receptors is rapid, reversible, specific and saturable. Scatchard analysis of the binding data indicates the presence of 116-275 identical receptors per neutrophil with an apparent dissociation constant (KD)of 1,l-2,3 nM. Only one class of binding sites was identified. The LTD, receptors are located in the plasma membrane and are specific for LTD, since binding is unaffected by other leukotrienes. Furthermore, LTD, induces a rapid and persistent translocation of Protein Kinase C (PKC) from the cytosol to the membrane. The LTD, binding and PKC translocation could be blocked in a concentration dependent manner by the new and specific LTD, receptor antagonist ICI 198,615. These observations strongly suggest that human PMN might possess specific LTD, receptors which are coupled to the inositol trisphosphate pathway resulting in a rise in the cytoplasmic free Ca2+and redistribution of protein kinase C. A mechanism of signal transduction for leukotriene D, is proposed.
Key words: Leukotriene receptors; inflammation; intracellular free Ca2'; ICI 198,615; second messengers; inositol trisphosphate; protein kinase C. Reprints: Pierre N. Bouchelouche, M.D., Dept. of Clinical Chemistry, KAS Herlev, Herlev Ringvej 75, DK-2730 Herlev, Denmark.
48 P. N. Bouchelouche & D. Beriki
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
INTRODUCTION Arachidonic acid metabolites may generate a variety of hypersensitivity and inflammation reactions. The cysteinyl leukotrienes (LTC,, LTD,, and LTE,) represent one family of arachidonic acid metabolites with potent effects in many organs and tissues [ 11. Leukotriene D, (LTD,) is a potent bronchoconstrictor and has primarily been studied in regard to the human airway [2]. In addition to its spasmogenic effects, LTD, promotes mucus secretion, and the clinical interest in the cysteinyl leukotrienes in obstructive airway diseases is evidently great [2]. Some tissues have already been described regarding receptors for leukotriene D, including tissue from guinea-pig [3], human lungs [4], and rat basophilic leukemia cells [5], while their existence in human blood leukocytes has never been reported [6]. Recently, our team provided indirect evidence of the existence of specific LTD, receptors in human PMN in which activation led to rapid breakdown of inositol lipids and increase in the intracellular concentration of free Ca" [6]. Using radioligand binding assays for ['HILTD, and selective LTD, antagonists, we have studied the binding characteristics of [3H]-LTD, to humans PMNs. The results obtained correspond to the kinetic expected for membrane associated receptors probably using PI turnover as part of the signal transduction.
Groningen, The Netherlands). Purified human albumin (Behring Werke, Marburg, FRG). SR2640 (Leo Pharmaceutical, Ballerup, Denmark). Fura-2/acetoxymethylester (fura-2/AM) and the non-ionic detergent Pluronic F-127 were obtained from Molecular Probes (Junction City, OR, USA). Ethyieneglycol-bis-(b-aminoethy1ether)N,N,N,N-tetraacetic acid (EGTA), diethylenetriamine pentaacetic acid (DTPA), digitonin (stock solution 10 g/L), CaCl,, leupeptin, dithiothreitol, PMSF, trypsin inhibitor, benzamidine and triton X-100 were obtained from Sigma Chemical Company ( S t . Louis, MO, USA). The purity and concentration of EGTA was determined by the ammonium oxalate method [7] and by titration with Ca2' [8], respectively. [y-3zP]-ATPwas from D U PONT Denmark A/S. RPMI-1640 from Flow Laboratories, Scotland. ICI 198,615 was a gift from ICI Pharmaceuticals, Wilmington, Delaware, USA. Isolation of neutrophils Venous blood (EDTA 10 mM) obtained from the cubital vein of healthy volunteers who had taken no drugs for at least 2 weeks was sedimented on methylcellulose and buffy coat leucocytes were fractionated by gradient centrifugation on lymphoprep after Boyum [9]. Erythrocytes were removed by hypotonlysis. The final suspension contained > 97 % neutrophils with a viability > 95 % judged by the trypan blue extrusion method. The leukocytes were kept at 4 "C and used within one hour.
MATERIALS AND METHODS Reagents ['HI-LTD, at a specific radioactivity of 106 Ci/mmol to 225 Ci/mmol (Amersham International, Denmark). LTB,, LTC,, LTD,, LTE, (Paesel GmbH, Frankfurt am Main, FRG). Ethylenediaminetetraacetat(EDTA) (Merck), methylcellulose (Bie & Berntsen, Copenhagen, Denmark), Lymphoprep (Nygaard, Oslo, Norway). Dibutylphthalat and dinonylphthalat (Merck). Instagel (Packard,
Receptor studies A radioligand binding assay similar to that previously described for LTB, receptors [lo, 111was used. Duplicate samples of PMN in 200 pL Gey buffer were incubated with rH]-LTD, (0.1-2.5 nM) with or without a 1000-fold excess of LTD, to determine nonspecific binding. Samples of lo7 cells/mL were incubated for 60 minutes at 25 "C with intermittent shaking. Following incubation the suspension was rapidly centrifuged
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
Signal transduction of LTD, in human neutrophils.
through a precooled dibutyl/dinonylphthalat oil layer (3/1 v/v) for 30 s at 11000 g. The bottom of the tube with the PMN were cut off and radioactivity was determined in a Tracor Analytic Scintillation Counter with automatic quench correction. The existence of multiple binding sites was evaluated by using a fixed concentration of 2.5 nM [3H]LTD, and varying concentrations of unlabelled LTD, (0.1- 1 pM). The specificity of the binding sites was investigated using structural LTD, analogues (LTB,, LTC,, LTE,) and specific LTD, receptor antagonists. The existence of intracellular binding sites was elaborated in experiments on sonicated and saponin treated cells. Fluorometric measurements of intracellular free calcium Cytosolic free CaZ+was measured with the calcium sensitive fluorescent dye fura-2. Fura-2 ester was kept in stock solution (dimethylsulfoxide) and diluted in RPMI1640 medium immediately before use. In order to improve dye loading fura-2/AM was added briefly sonicated to obtain fine emulsions and an equal volume of the nonionic detergent Pluronic F-127 (25 % w/v) was added. Briefly described the cells (5.lo6cells/mL) were loaded by incubation for 30 min at 37 "C with 1 pM of the fura-2/AM mixture, washed twice to remove the extracellular dye and left at room temperature until use. The medium for fluorescence measurements consisted of (in mM): 145 NaCI, 5 KCl, 1 Na,HPO,, 1 CaCl,, 0.5 MgSO,, 5 glucose and 20 HEPES buffer. The medium was adjusted with NaOH to pH 7.4 at 37 "C. Time course of change in fluorescence was measured either by single (340 nm) or dual (340 and 380 nm) excitation wavelength spectrometry as previously described [ 12,131. The photometer was programmed to scan back and forth between the two excitation wavelengths and to reject data during the transition period. This technique is more cumbersome and was therefore only used in those experiments to determine the absolute
49
concentration of intracellular free Ca". The advantages of the dual-wavelength method have already been described [14]. Autofluorescence values were obtained from matching suspensions of unloaded cells from the same batch. The dissociation constant of the Caz'-fura-2 complex was assumed to be 224 nM [15]. None of the experimental agents used altered the autofluorescence of the cells. Protein kinase C translocation assay Buffer solutions: Buffer A contained (in mM): 20 TRIS-HCI, 10 EGTA, 4 EDTA and 157 mg/L benzamidine. pH was adjusted to 7.5. Buffer B contained (in mM): 20 TRIS-HCl, 5 EGTA, 2 EDTA and 157 mg/L benzamidine. Buffer C contained (in mM): 20 TRIS-HCL, 0.5 EGTA, 0.5 EDTA and 157 mg benzamidine. At the day of the experiment trypsin inhibitor (1 mg/mL), dithiothreitol (1 mM), PMSF (20 pL/mL) and leupeptin (3pLlmL) were added to all buffers. Intact human polymorphonuclear leukocytes (5.10' cells/mL) were preincubated in 3 mL RPMI-1640 with or without LTD, (lo9- lo' M). The reactions were stopped with 10 mL ice-cold RPMI-1640 medium at the time indicated in the figures. The cells were rapidly centrifugated at 300 g for 5 min. The cell pellets were resuspended in 2 mL buffer A and ultrasonically disrupted at 4 "C. The crude homogenates were centrifuged at 100.000 g for 60 min at 4 "C. After centrifugation, 2 mL buffer A containing 0.1 % triton X-100 was added to the supernatant (cytosol fraction). The pellet (membrane fraction) was resuspended in 4 mL buffer containing 0.1 % triton X-100, stored on ice for one hour and finally centrifuged at 100.000 g for 30 min. Membrane and cytosol fractions were chromatographed in parallel on DEAE-cellulose columns (1.4 cm) using an FPLC system (Pharmacia, Uppsala, Sweden). The columns were preequilibrated with 20 mL buffer A. There-
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
50 P. N. Bouchelouche & D. Berild after, the columns were washed with 10 mL buffer B, 10 mL buffer C and finally with 3 mL buffer C containing 25 mM NaC1. Protein kinase C and the phospholipidindependent PKC were eluted with 5 mL buffer C containing 100 and 400 mM NaCl, respectively. PKC activity was determinated by measurement of the transfer of "P from [y"P]ATF' to histone [16]. The reaction was stopped after 10 min at 30 "C with ice-cold ATP in excess and an aliquot of the mixture applied onto 3.3 cm' squares of Whatman P-81 chromatography paper. Following thorough washing in 50 mM NaCl and overnight drying radioactivity was determinated by scintillation counting. PKC activity was defined as the difference between the 32P incorporation in the presence and absence of phosphatidylserine and diolein [ 171.
FIG. 1. Association and dissociation curve for the specific binding of ['HI-LTD, to human PMN leucocytes. 2.5 nM [ HI-LTD, was added to lo7 PMN/mL and bound radioactivity was measured every 10 min. After 55 min excess of LTD, was added to the suspension and dissociation was measured. The curve is corrected for non-specific binding as assessed by addition of 2.5 pM unlabelled LTD,.
RESULTS
density (Bmax) of 116-275 binding sites per cell (n=3).
Optimal conditions for receptor binding studies were established at a cell concentration of lo7 PMN/mL and incubation at 25 "C for 60 min in a Gey buffer containing 0.1 % bovine albumin. Characterization of [)H]-LTD, binding to human PMN leukocytes The time course for binding of ['HI-LTD, to human PMNs is shown in Fig. 1. This figure also demonstrates the rapid reversibility caused by addition of excess unlabelled LTD,. Fig. 2A demonstrates a typical saturation curve for binding of [3H]-LTD,(0.1-2.0 nM) to PMN. Non-specific binding was linear with increasing concentrations of ['HI-LTD, (data not shown). In competition experiments using fixed concentrations of 2.5 nM ['HI-LTD, and varying concentrations of unlabelled LTD, (0.1 - 1 pM) no further binding sites were detected. Kinetic analysis using the Scatchard plot (Fig. 2B) revealed a dissociation constant (K,) of 1.1-2.3 nM with a receptor
-
1.01 .o
I
r
9 m 0 505
I
0
1
30
60
120 lio
Time (rnin)
Inhibition of [.'H]-LTD, binding to human PMNs by structurally related leukotrienes and LTD, receptor antagonists In competition experiments unlabelled LTD, inhibited [3H]-LTD,binding in a concentration dependent manner (Fig. 3). The structurally related leukotrienes LIB, and LTE4 did not influence LTD4binding, while LTC, inhibited at concentrations above 10" M. Fig. 4A shows that the ['HI-LTD, binding is inhibited in a concentration dependent manner by the specific LTD, receptor antagonist SR2640, while Fig. 4B demonstrates inhibition by the more potent LTD, receptor antagonist ICI 198,615 [18] resulting in approximately 90 % inhibition at a concentration of only 0.1 nM. LTD, receptor-mediated cytosolic free Ca" mobilization Human PMN (5.106cells/mL) loaded with fura-2 were suspended in calcium rich medium (left) and in calcium free medium (middle). LTD, was added as indicated. In Fig. 5 (right), human PMN was incubated
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
Signal transduction of LTD, in human neutrophils. 5 1
Concentration of LTDq (nM) FIG.2. A. Concentration dependence of the specific binding of ['HI-LTD, to human PMN leukocytes. lo7 PMN/mL were incubated with increasing concentrations of ['HI-LTD, at 25" for 60 min. The curve is corrected for nonspecific binding as assessed by addition of 2.5 gM unlabelled LTD,. B. Typical Scatchard plot derived from binding in Fig. 2.4.
(5 min) with the LTD, receptor-antagonist ICI 198,615 and then stimulated with LTD, as indicated. Typical trace of 10 experiments.
1.5
1.0.
r r L
Q 0.5
I
10
9
8
7
6
Concentration(.lqM)
FIG. 3 Competitive inhibition of ['HI-LTD, to human leukocytes by structurally related hg&ds. Suspensions of lo' PMN/mL were incubated at 25" for 60 min with 2 nM ['HI-LTD,. Non-radioactive LTB,, LTC,, LTD, and LTE, were added to the cell suspensions in concentration 10.'' M to lod M.
Protein kinase C activation. To investigate whether the stimulation of the LTD,-receptor was linked to the phospholipids-sensitiveCa*+-dependentprotein kinase C, we studied the translocation of this enzyme in the particulate and the cytosol fraction of human PMN treated with LTD,. PKC redistribution from cytosol to membrane is usually accepted as the first step of its activation [19]. As can be seen from Fig. 6.4, stimulation of the cells with LTD, provokes a rapid and marked reduction in the PKC activity in the cytosol and an increase in the membrane fraction. The translocation of PKC started immediately after addition of LTD, and reached a maximum within 5-10 min. Preincubation of the cells with 10 nM of the receptor antagonist ICI 198,615 com-
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
52 P. N. Bouchelouche & D.Berild
0
I
I
10
100
0
f
1Mxl
0
loo00
Concentration of SRZMO (nM)
01
02
03
04
Concentration of ICI 198,615 (nM)
FIG. 4. A. The influence of the specific LTD, receptor anta onist SR2640 on the binding of I3H]-LTD, to human PMN leucocytes. lo7 PMN/mL was incubated with 2.5 nM [5HI-LTD,. After equilibrium was achieved, increasing concentrations of the antagonist were added and the bound radioactivity measured. B. The influence of the more potent LTD, receptor antagonist ICI 198,615 on the percentage [’HI-LTD, bound to human PMN at equilibrium. The procedure was as in Fig. 4A.
[
); , : : ,
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-6M
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10 nM
10 nM
LTDI
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LTOi
FIG.5. LTD,-induced increase in PMN cytosolic free Ca”. Leff and middle: Intact human PMN loaded with fura-2 were suspended in medium containing 1 mM calcium and in calcium free medium, respectively. The cells were challenged with 10 nh4 LTD, and fluorescence was recorded. Right: Effect of the LTD, receptor antagonist ICI 198,615 on the maximum increase in [Ca”],. Each trace is representative of 3 separate experiments.
pletely abolished the translocation of the enzyme (Fig. 6B) DISCUSSION The possibility that human PMN leukocytes might express receptors for LTD, was initially suggested in our previous report on
LTD, induced increase in the level of intracellular free Ca2+,generation of inositol phosphates and down regulation of the human PMN migration response [6]. The finding that the LTD, receptor antagonist _ . . . ._. . - . . . SKZ640 is able to inhibit the Ll‘U,-induced generation of these second messengers and to suppress the attenuation by LTD, of LTB,-directed PMN chemotaxis [6], indicates that LTD,-receptors were involved. The presence of specific receptors for leukotriene D, in a number of tissues including human [4] and guinea pig lungs [3], human alveolar macrophages [20], and rat basophilic leukemia cells [5] has been demonstrated in a number of studies. Moreover, Bach et a1 have recently obtained preliminary evidence documenting the presence of specific LID, binding sites on cultured mesangial cells [21]. In the present study we used chemical and pharmacological methods to identify LTD, binding sites on human PMN, and it is shown for the first time that human PMNs might possess specific LTD, receptors at their plasma membrane. The binding is reversible and shows rapid saturation and a
Signal transduction of LTD, in human neutrophils. 53
Membrane
-0
lO?y
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
-6
-
cytosol
20
10
30
Time (min)
Time (min)
FIG. 6. A. Effect of LTD, on PKC redistribution from cytosol to membrane. PKC activity was determined in cytosolic and membrane fractions of human PMN treated with 0.1 pM LTD, at indicated times. Representative trace
of 2 experiments each assayed in triplicate. B. Effect of the LTD, receptor antagonist ICI 198,615 (10 nM)on PKC activity in LTD, stimulated human PMN. Representative trace of 3 experiments in triplicate.
FIG. 7. Model for signal transduction mechanism for
LTD, in human PMN leukocytes.
high affinity of ['HI-LTD, for the binding sites. The specificity of the LTD, receptors is supported by the lack of effect of LTB, and LTE, on ['HI-LTD, binding and the competitive inhibition by LTD, and by the LTD, receptor antagonists ICI 198,615 and SR2640. The enzymatic conversion of LTC, into LTD, may explain the small competitive inhibition of binding of ['HI-LTD, by LTC,. This could be due to the fact that PMN leukocytes are capable of both synthesis and degradation of LTC, [22]. Interaction of LTC, with y-glutamyltranspeptidase results in cleavage of glutamic acid and glycine to yield LTD, [23]. Permeabilization of the cells with the nonionic detergent saponin or disruption of the cells by sonication did not increase the number of binding sites, indicating that human PMN leukocytes possess no LTD,
receptors bound to intracellular organelles in contrast to LTC, [22]. Furthermore, the PMN leukocyte receptor for LTD, differs from those of LTB, with respect to affinity. The LTB, receptor consists of both high and low affinity receptors [ 101. Scatchard analysis of the specific binding of LTD, to the cells in this study revealed a single class of binding sites with very few receptors per cell compared to LTB, [lo]. Addition of LTD, to human PMN causes an immediate and transient increase in [Ca'+], in Ca rich as well as in Ca free media. The threshold concentration for effect was 10-l' M LTD, and maximal response was achieved at 10.' M LTD,. Furthermore, LTD, causes a translocation in PKC from the cytosol to the membrane in the concentration range 10"o-lO' M LTD,. The observation that the response in [Ca"], to LTD, can be obtained at concentrations very similar to the &-value for interaction between 'LTD, and its receptor, in addition to the inhibition of both receptor binding and response by the specific LTD, receptor antagonist ICI 198,615 strongly indicate that CaZt mobilization and PKC activation are mediated through binding of LTD, to the receptor. Previously, we have demonstrated that stimulation of human PMN with LTD4 resulted in the breakdown of phosphatidylinositoi bisphosphate. Therefore, it seems
Scandinavian Journal of Clinical and Laboratory Investigation 1991.51:47-55.
54 P. N. Bouchelouche & D. Berild
likely that in human PMNs the binding of LTD, to its receptor results in the formation of IP,, which triggers the release of Ca2+ from intracellular stores, and translocation of protein kinase C. In conclusion, the simplest tentative model of the signal transduction system of the leukotriene D, receptor is illustrated in Fig. 7. LTD, binding to the receptor activates phospholipase C (PLC)at the plasma membrane. Activation of this enzyme leads to cleavage of phosphatidylbisphosphate(PIP,), resulting in the formation of two second messengers inositol 1,4,5-trisphosphate (IP,) and diacylglycerol (DG). Inositol 1,4,5trisphosphate mobilizes calcium from internal stores resulting in transient increase in the cytosolic free Ca2+,while diacylglycerol, ilicreases protein phosphorylation by activation of protein kinase C (PKC).
4.
5.
6.
I. 8.
9.
10.
ACKNOWLEDGEMENTS We wish to thank Mrs. Annie Petersen for her skilled assistance in the binding study experiments. We also thank ICI Pharmaceuticals Products, Wilmington, Delaware, USA for the kind gift of ICI 198,615 and Leo Pharmaceuticals Products, Denmark for the supply of SR2640. We further thank N.O. Christiansen and C.S. Larsen (Department of Medicine and Infectious Diseases, Marselisborg Hospital, k h u s C, Denmark) for familiarizing us with the field of protein kinase C.
11.
12.
13.
14.
15.
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Lewis MA, Mong S, Vessella RL, Crooke ST. Identification and characterization of leukotriene D, receptors in adult and fetal human lung. Biochem Pharmacol 1985; 34: 4311-7. Mong S, Wu HL, Wong A, Saran HM, Crooke ST. Leukotriene D4 receptor-mediated phosphoinositol hydrolysis and calcium mobilization in rat basophilic leukemic cells. J Pharmacol Exp Ther 1988, 247 803-13. Bouchelouche PN, Ahnfelt-RBnne I, Thomsen MK. LTD, increases cytosolic free calcium and inositol phosphates in human neutrophils: Inhibition by the novel LTD, receptor antagonist, SR2640, and possible relation to modulation of chemotaxis. Agents and Actions 1990; 29: 299307. Miller DJ, Smith GL. EGTA purity and the buffering of calcium ions in physiological solutions. Am J Physiol 1984; 246: C160-6. Moisescu DG, Push H. A pHmetric method for the determination of the relative concentration of calcium to EGTA (abstr.). Pfliigers Arch 1875; 355 R122. Boyum A. Separation of blood leucocytes, granulocytes and lymphocytes. Tissue Antigens 1973; 4 269-74. Goldman BW, Goetzl El. Heterogeneity of human polymorphonuclear leukocyte receptors for leukotriene B,. Identification of a subset of high affinity receptors that transduce the chemotactic response. J Exp Med 1984; 159 1027-41. Berild D, Bouchelouche PN, Skovgaard-Poulsen H, Elmgren J. Impaired cytosolic free Ca2+ response, chemotaxis and leukotriene B, receptor function in peripheral neutrophils from patients with Crohn's disease. Submitted for publication. Bouchelouche PN, Hainau B, Frederiksen 0. Effect of BAY K 8644 on cytosolic free calcium in isolated rabbit gall-bladder epithelial cells. Cell Calcium 1989; 10: 37-46. Bouchelouche PN, Reimert C, Bendtzen K. Effects of natural and recombinant interleukin la and -B on cytosolic free calcium in human and murine fibroblasts. Leukemia 1988,2 691-6. Tsien RY, Rink TJ, Poenie M. Measurement of of cytosolic free Ca'' in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 1985; 6: 145-53. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260:3440-50, Christiansen NO, Larsen CS, Juhl H, Esmann V. Membrane-associated protein b a s e s in phorbol ester-activated human polymorphonuclear leukocytes. Biochem Biophys Acta 1986; 884: 54-9. Su HD, Shoji M, Mazzei GJ, Vogler WR,Kuo JF. Effects of selenium compounds on phospholipids/Ca,+-dependent protein kinase C (protein kinase C) system from human leukemic cells. Cancer Research 1986; 46: 3684-7.
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Signal transduction of LTD, in human neutrophils. 55 18. Snyder DW, Gdes RE, Keith RA,Yee YK, Krell RD. The in vifro pharmacology of ICI 198,615: A novel, potent and selective peptide leukotriene antagonist. J Pharmacol Exp Ther 1987; 243 548-56. 19. Turner RS, Kuo JF. Phospholipids-sensitive Ca” -dependent protein b a s e C (protein kinase C): The enzyme, substrates, and regulation. In: Kuo JF (ed). Phospholipids and cellular regulation, vol 11. CRC Press, 1986. 20. Opmeer FA, Hoogsteden HC. Characterization of specific receptors for leukotriene D, on human alveolar macrophages. Prostaglandins 1984; 28. 183-95. 21. Bard KF, Ebert J, Hoover RL, Mong S, Harris RC. Characterization of leukotriene D, binding to rat glomerular mesanglial cells: demonstration of LTD,-stimulated inositol phosphate synthesis and ’H-thymidine incorporation. Kidney Int 1988, 3: 253 (abstr). 22. Baud L, Koo CH, Goetzl El. Specifity and cellular distribution of human polymorphonuclear leukocyte receptors for leukotriene C,. Immunology 1987; 6 2 53-9. 23. Krilis S, Lewis RA, Corey El, Austen FK. Bioconversion of C-6 sulfidopeptide leukotrienes by the responding guinea pig ileum determines the time course of its contraction. J Clh Invest 1983: 71: 909-15.
