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Research Paper
Mediators of Inflammation, 2, 149-151 (1993)
THE effect of PAF on the plasma membrane polarity ofpolymorphonuclear leukocytes (PMNs) was investigatedby measuring the steady-state fluorescence emissionspectra of 2-dimethylamino(6-1auroyl) naphthalene(Laurdan), which is known to be incorporated at thehydrophobic-hydrophilic interface of the bilayer, dis-playing spectral sensitivity to the polarity of itssurrounding. Laurdan shows a marked steady-stateemission blue-shift in non-polar solvents, with respect to
polar solvents. Our results demonstrate that PAF (10-7 M)induces a blue shift of the fluorescence emission spectraof Laurdan. These changes are blocked in the presence ofthe PAF antagonist, L-659,989. Our data indicate that theinteraction between PAF and PMNs is accompanied by adecrease in polarity in the hydrophobic-hydrophilicinterface of the plasma membrane.
Key words: Laurdan, PAF, Plasma membrane, Poly-morphonuclear leukocytes
Effect of PAF onpolyrnorphonuclear leucocyteplasma membrane polarity" afluorescence study
A. Kantar,1"cA P. L. Giorgi, and R. Fiorini2
Departments of Pediatrics and 2Biochemistry,University of Ancona, via Corridoni 1,60123Ancona, Italy
CA Corresponding Author
Introduction
Platelet-activating factor (PAF, 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine) is a potentlipid mediator with a diverse spectrum of biologicalactivities in a variety of cells. 1’2 These activities are
3due to the interaction of PAF with its receptors.Human polymorphonuclear leukocytes (PMNs)plasma membrane possess a cell surface receptorthat specifically binds PAF and initiates variousbiochemical responses to PAF.3’4 Several studieshave provided information on the nature of theinteractions of PAF with PMNs membrane receptorand the signalling pathways involved in its cellularaction. 3-5
Fluorescence spectroscopy techniques have beenwidely used to study the physico-chemical changesof membrane organization. 6-8 The fluorescentmembrane probe Laurdan (2-dimethylamino(6-lauroyl) naphthalene) is known to be sensitive to
the polarity of the environment, displaying spectralsensitivity to the phospholipid phase state. In thephospholipid’s liquid-crystalline phase and withinthe phase transition, Laurdan displays a red-shiftedspectrum. The red shift has been attributed to
relaxation processes that involve motions of thephospholipid polar head groups when they undergoreorientation around the excited state of the probe.On the other hand, in the gel phase the decrease ofmotional freedom of the bilayer polar residuesresults in an unrelaxed blue-shift spectrum.9
In this study we have investigated the effect ofPAF on membrane polarity of PMNs by measuringsteady-state emission spectra of Laurdan in-corporated into the PMNs plasma membrane beforeand after addition ofPAF in the absence or presence
(C) 1993 Rapid Communications of Oxford Ltd
of the PAF antagonist1 (+/-)-trans-2-(3-methoxy-5-methylsulphonyl 4 propoxyphenyl) 5 (3,4,5 trimethyloxyphenyl) tetrahydrofuran (L-659,989).
Materials and Methods
Preparation of PMNs: Human PMNs were isolatedfrom freshly drawn blood of ten healthy volunteersusing a Mono-Poly Resolving Medium (ICNBiomedicals, Milan, Italy) as previously described. 11
Cells were suspended in Krebs-Ringer phosphatesolution supplemented with 1 mg/ml glucose at a
final concentration of 106 PMNs/ml for chemilumi-nescence and fluorescence studies.
Chemiluminescence measurements: Luminol amplifiedchemiluminescence was measured in an AutoLumatLB 953 (Berthold Co, Wilbad, Germany) and PMNs(in the absence or presence of Laurdan) wereactivated by addition of PAF (Sigma Chemical Co.,St Louis, MO, USA) (5 x 10 -8 M) as previouslydescribed. 12
Fluorescence measurements: PMNs were labelled with anethanol solution of Laurdan (Molecular Probes Inc.Eugene, OR, USA) under N2 at a final probeconcentration of 0.05/M as previously described.3
Samples were prepared at room temperature in redlight and used immediately for fluorescencemeasurements. Steady-state excitation and emission
spectra were measured at 37C before and afteraddition of PAF on a photon counting spec-trofluoromete (model GREG PC, ISS Inc.,Urbana, IL, USA) and using the ISS Inc. software.The actual temperature was measured in the samplecuvette using a digital thermometer. PAF was
Mediators of Inflammation Vol 2.1993 149
A. Kantar, P. L. Giorgi and R. Fiorini
added to PMNs at a final concentration of 10 -v M.The PAF antagonist, L-659,989 (kindly donated byDr W. H. Parsons, Merck Sharp & DohmeResearch Laboratories, NJ, USA) was added toPMNs at a final concentration of 10 -6 M.l
Results
Chemiluminescence studies: Luminol amplified chemilu-minescence has been employed, as previouslydescribed, 12’13 to verify that in isolated PMNs thesuperoxide-generating oxidase system is dormantunder basal conditions and can be activated by PAF.All samples, used in this study, demonstrated anactivable NADPH-oxidase system (data not shown).PMNs labelled with Laurdan 0.05 #M still showeda normal oxidative burst.
Fluorescence studies: The background phospholipidfluorescence of PMNs was checked prior to eachmeasurement and was less than 0.1% of thefluorescence when Laurdan was added. Thecontribution of the light scattering was negligiblein our samples because of the low cell concentrationused in this study. The fluorescence intensity of thefree probe in KRP, in the absence of PMNs, was
negligible and did not increase upon the addition ofPAF at the concentrations used in the study;moreover, this concentration (10 -v M) is lowerthan the critical micelle concentration of PAF2 x 10 -s M as reported in literature. TM The kineticof incorporation of Laurdan (0.05/iM) in PMNsplasma membranes was established as recentlydescribed. 13
Fluorescence emission and excitation spectra ofLaurdan in PMNs before and after addition of PAF10-VM are reported in Figs 1 and 2, respectively.Figure 1 shows emission spectra measured at anexcitation wavelength of 350 nm. After the addition
1.00
0.75
508 550WAVE LENGTH (nm)
FIG. 1. Normalized emission spectra of Laurdan incorporated in PMNsplasma membrane at 37C before () and after (mOrn) addition ofPAF 10-7 M. The spectra were measured at an excitation wavelength of350 nm.
1 50 Mediators of Inflammation. Vol 2.1993
1.00
0.75
395 420WAVELENGTH
FIG. 2. Normalized excitation spectra of Laurdan incorporated in PMNsplasma membrane at 37C before () and after (mOrn) addition ofPAF 10-7 M. The spectra were measured at an emission wavelength of450 nm.
I- 0.50
Z114
Z0.26
0320 348 370
of PAF a 15 nm blue-shift of the Laurdan emissionspectrum was observed. The emission maximum inbasal conditions (unstimulated PMNs) was at
443nm, while the emission maximum afterstimulation with PAF was at 428 nm. Figure 2shows Laurdan excitation spectra measured at anemission wavelength of 450 nm. The addition ofPAF induced a decrease in intensity at longerwavelengths with respect to basal conditions.
In the presence of L-659,989, no significantchanges in the emission or excitation spectra wereobserved after the addition of PAF.
Discussion
The fluorescent probe Laurdan has been reportedto be incorporated at the hydrophilic-hydrophobicinterface of the membraneis with the lauric acid tailanchored in the hydrophobic region of the bilayer.It has been demonstrated that Laurdan displaysspectral sensitivity to the polarity of its surround-ing, showing a red-shift of the emission in polarsolvents, with respect to non-polar solvents.9 Thisbehaviour is referred to dipolar relaxation pheno-mena, that are related to the physical state and thedynamics of the surrounding phospholipid polarhead group.9’16 In single-phase phospholipidvesicles the dynamics of the surroundings detectedby Laurdan is very different in the case of the gelor of the liquid-crystalline phase; the probe showsa marked steady-state emission red-shift in thephospholipid liquid-crystalline phase, with respectto the gel phase.9 If solvent molecules can moveduring the fluorescence lifetime, the Laurdanexcited-state molecular dipole will orient theneighbouring solvent dipoles, and the steady-stateLaurdan emission spectrum will be red-shifted, iv
Our results demonstrate that PAF induced ablue-shift of the emission spectra of Laurdan
PAF effects on leucocyte plasma membrane polarity
incorporated in the plasma membrane, indicating adecrease in polarity of the environment surround-ing the probe. In basal conditions, Laurdanemission reflects the interactions between the probeand the phospholipid polar head groups. Theaddition of PAF to PMNs decreases the motionalfreedom of the bilayer polar residues, as demon-strated by the presence of an unrelaxed blue-shiftof the spectrum. In previous studies, 12’18 using thefluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-l,3,5-hexatriene (TMA-DPH), we haveshown that PAF 10-7M induces an increase inPMNs membrane phospholipid packing and adecrease in membrane microheterogeneity. Thesephysico-chemical changes may cause a reduction ofwater penetration in the hydrophobic environmentsurrounding the Laurdan moleculeS, thus inducinga decrease in polarity. Because these changes in thephysico-chemical organization of the membrane canbe blocked by the PAF antagonist L-659,989, itseems likely that they are attributed to PAF-receptor interaction and the biochemical events
associated with signal transduction.
References1. Koltai M, Hosford D, Guinot P, Esanu A, Braquet P. Platelet activating
factor (PAF): review of its effects, antagonists and possible future clinicalimplications (Part I). Drugs 1991; 42: 9-29.
2. Koltai M, Hosford D, Guinot P, Esanu A, Braqet P. PAF: review of itseffects, antagonists and possible future clinical implications (Part II). Drugs1991; 42: 174-204.
3. Shukla SD. Platelet activating factor receptor and signal transductionmechanisms. FASEB J 1992; 6: 2296-2301.
4. Hwang S-B. Specific receptors of platelet-activating factor, receptorheterogeneity, and signal transduction mechanisms. J Lipid Mediators 1990;2: 123-158.
5. Nakamura M, Honda Z, Izumi T, et al. Molecular cloning and expressionof platelet activating factor receptor from human leukocytes. J Biol Chem1991 266: 20400-20405.
6. I.akowicz JR. Fluorescence polarization. In: I,akowicz JR, ed. Principles ofFluorescence Spectroscopy. New York: Plenum Press, 1983; 709-733.
7. Fiorini R, Gratton E, Curatola G. Effect of cholesterol membranemicroheterogeneity: study using 1,6-diphenyl-l,3,5-hexatriene fluorescencelifetime distribution. Biochim Biophys Acta 1989; 1006: 198-202.
8. Kantar A, Giorgi PL, Curatola.G, Fiorini R. Effect of PAF erythocytemembrane heterogeneity: fluorescence study. Agents & Actions 1991; 32:347-350.
9. Parasassi T, Conti F, Gratton E. Time-resolved fluorescence emission spectraof Laurdan in phospholipid vesicles by multifrequency phase and modulation
fluorometry. Cell Biol 1986; 32: 103-108.10. Ponpipom MM, Hwang S-B, Doebber TW, et al. (___)-trans-2-(3-Methoxy-
methylsulfonyl 4 propoxyphenyl) trimethoxyphenyltetrahydrofuran(L-659,989), novel, potent PAF receptor antagonist. Biochem Biophys ResCommun 1988; 150: 1213-1220.
11. Kantar A, Wilkins G, Swoboda B, et al. Alterations of the respiratory burstof polymorphonuclear leukocytes from diabetic children: chemilumi-
study. A cta Paediatr Scand 1990; 29: 535-541.12. Fiorini R, Curatola G, Bertoli E, Giorgi PL, Kantar A. Changes of
fluorescence anisotropy in plasma membrane of human polymorphonuclearleukocytes during the respiratory burst phenomenon. FEBS Lett 1990; 223:122-126.
13. Fiorini R, Curatola G, Kantar A, Giorgi PL, Gratton E. Use of Laurdanfluorescence in studying plasma membrane organization of polymorpho-nuclear leukocytes during the respiratory burst. Photochem PhotoBio11993; 5"/:
438-441.14. Varveri FS, Mantaka-Marketou AE, Papadopoulos K, Nikokavouras J.
Chemiluminescence in organized molecular assemblies: chemiluminescenceof lucigenin in Lyso-PAF (C1). J Photochem Photobiol 1992; 66: 113-118.
15. Chong PLG. EFfects of hydrostatic pressure the location of Prodan in
lipid bilayers and cellular membranes. Biochemistry 1988; 22: 399-404.16. Parasassi T, Conti F, Gratton E. Fluorophores in polar medium: time
dependence of emission spectra detected by multifrequency phase andmodulation fluorometry. Cell Mol Biol 1986; 32: 99-102.
17. Parasassi T, De Stasio G, D’Ubaldo A, Gratton E. Phase fluctuation inphospholipid membranes revealed by Laurdan fluorescence. Biophys J 1990;57: 1179-1186.
18. Kantar A, Giorgi PL, Curatola G, Fiorini R. Changes in plasma membranemicroheterogeneity of polymorphonuclear leukocytes during the activationof the respiratory burst. In: Jesaitis Aj, Dratz EA, eds. Molecular basis ofoxidative damage by leukocytes. Boca Raton: CRC Press, 1992; 243-246.
ACKNOWLEDGEMENTS. Part of this work performed in the Laboratoryfor Fluorescence Dynamics (LFD) at the University of Illinois at
Urbana-Champaign (UIUC). The LFD is supported jointly by the Division ofResearch Resources of the NIH and UIUC. We indebted to Prof. EnricoGratton for his kind assistance.
Received 6 January 1993"accepted in revised form 15 February 1993
Mediators of Inflammation. Vol 2.1993 151
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