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• W1990247966 abstract "Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform. Some isoforms of secretory phospholipase A2 (sPLA2) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA2 isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6–48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA2 were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform. Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.Journal of Biological ChemistryVol. 285Issue 26PreviewVOLUME 285 (2010) PAGES 10993–11002 Full-Text PDF Open Access During programmed cell death, or apoptosis, a variety of changes occur in the plasma membrane of the cell. These include morphological alterations that emerge late in the process such as blebbing and increased permeability of the membrane. Earlier in the process, several more subtle membrane changes occur. The best studied is a loss of the normal asymmetrical transmembrane distribution of phospholipid species. Consequently, anionic lipids like phosphatidylserine, which are typically confined to the inner leaflet of the membrane, become exposed on the outer surface (1.Fadeel B. Antioxid. Redox Signal. 2004; 6: 269-275Crossref PubMed Scopus (49) Google Scholar). In addition, studies with fluorescent membrane probes have revealed possible increases in fluidity and/or the spacing between lipid molecules that may precede or coincide with the loss of membrane asymmetry, depending on the cell type and mode of apoptosis (2.Mower Jr., D.A. Peckham D.W. Illera V.A. Fishbaugh J.K. Stunz L.L. Ashman R.F. J. Immunol. 1994; 152: 4832-4842PubMed Google Scholar, 3.Jourd'heuil D. Aspinall A. Reynolds J.D. Meddings J.B. Can. J. Physiol. Pharmacol. 1996; 74: 706-711Crossref PubMed Scopus (17) Google Scholar, 4.Fujimoto K. Iwasaki C. Kawaguchi H. Yasugi E. Oshima M. FEBS Lett. 1999; 446: 113-116Crossref PubMed Scopus (41) Google Scholar, 5.Raghavendra P.B. Sreenivasan Y. Manna S.K. Mol. Immunol. 2007; 44: 2292-2302Crossref PubMed Scopus (46) Google Scholar, 6.Baritaki S. Apostolakis S. Kanellou P. Dimanche-Boitrel M.T. Spandidos D.A. Bonavida B. Adv. Cancer Res. 2007; 98: 149-190Crossref PubMed Scopus (61) Google Scholar, 7.Moulin M. Carpentier S. Levade T. Arrigo A.P. Apoptosis. 2007; 12: 1703-1720Crossref PubMed Scopus (42) Google Scholar, 8.Bailey R.W. Olson E.D. Vu M.P. Brueseke T.J. Robertson L. Christensen R.E. Parker K.H. Judd A.M. Bell J.D. Biophys. J. 2007; 93: 2350-2362Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 9.Bailey R.W. Nguyen T. Robertson L. Gibbons E. Nelson J. Christensen R.E. Bell J.P. Judd A.M. Bell J.D. Biophys. J. 2009; 96: 2709-2718Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Recently, a latent increase in the order of membrane lipids has also been reported (9.Bailey R.W. Nguyen T. Robertson L. Gibbons E. Nelson J. Christensen R.E. Bell J.P. Judd A.M. Bell J.D. Biophys. J. 2009; 96: 2709-2718Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). A potential consequence of these events during apoptosis is enzymatic attack of the cell membrane by secretory phospholipase A2 (sPLA2). 2The abbreviations used are: sPLA2secretory phospholipase A2hGIIahuman group IIahGVhuman group VhGXhuman group XAppD49monomeric aspartate 49 sPLA2 from venom of A. p. piscivorusADIFABacrylodan-derivatized fatty acid-binding proteinMBSSmodified balanced salt solutionGPgeneralized polarization. Ordinarily, healthy cells resist hydrolysis, but during apoptosis they become vulnerable to destruction by the enzyme (9.Bailey R.W. Nguyen T. Robertson L. Gibbons E. Nelson J. Christensen R.E. Bell J.P. Judd A.M. Bell J.D. Biophys. J. 2009; 96: 2709-2718Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 10.Nielson K.H. Olsen C.A. Allred D.V. O'Neill K.L. Burton G.F. Bell J.D. Biochim. Biophys. Acta. 2000; 1484: 163-174Crossref PubMed Scopus (24) Google Scholar, 11.Atsumi G. Murakami M. Tajima M. Shimbara S. Hara N. Kudo I. Biochim. Biophys. Acta. 1997; 1349: 43-54Crossref PubMed Scopus (88) Google Scholar). Studies with snake venom phospholipase A2 have identified possible ways by which this phenomenon relates to membrane physical properties (8.Bailey R.W. Olson E.D. Vu M.P. Brueseke T.J. Robertson L. Christensen R.E. Parker K.H. Judd A.M. Bell J.D. Biophys. J. 2007; 93: 2350-2362Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 9.Bailey R.W. Nguyen T. Robertson L. Gibbons E. Nelson J. Christensen R.E. Bell J.P. Judd A.M. Bell J.D. Biophys. J. 2009; 96: 2709-2718Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 12.Jensen L.B. Burgess N.K. Gonda D.D. Spencer E. Wilson-Ashworth H.A. Driscoll E. Vu M.P. Fairbourn J.L. Judd A.M. Bell J.D. Biophys. J. 2005; 88: 2692-2705Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Preliminary investigations suggest that human groups IIa (hGIIa) and V (hGV) isoforms may also distinguish healthy and apoptotic cells, although the details of how they do so are uncertain (11.Atsumi G. Murakami M. Tajima M. Shimbara S. Hara N. Kudo I. Biochim. Biophys. Acta. 1997; 1349: 43-54Crossref PubMed Scopus (88) Google Scholar, 13.Wilson H.A. Waldrip J.B. Nielson K.H. Judd A.M. Han S.K. Cho W. Sims P.J. Bell J.D. J. Biol. Chem. 1999; 274: 11494-11504Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14.Smith S.K. Farnbach A.R. Harris F.M. Hawes A.C. Jackson L.R. Judd A.M. Vest R.S. Sanchez S. Bell J.D. J. Biol. Chem. 2001; 276: 22732-22741Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The response of the human group X (hGX) isozyme to apoptosis has not yet been studied. secretory phospholipase A2 human group IIa human group V human group X monomeric aspartate 49 sPLA2 from venom of A. p. piscivorus acrylodan-derivatized fatty acid-binding protein modified balanced salt solution generalized polarization. Hydrolysis of artificial membranes by sPLA2 involves two precatalytic steps (Scheme 1 (15.Henshaw J.B. Olsen C.A. Farnbach A.R. Nielson K.H. Bell J.D. Biochemistry. 1998; 37: 10709-10721Crossref PubMed Scopus (43) Google Scholar, 16.Gelb M.H. Jain M.K. Hanel A.M. Berg O.G. Annu. Rev. Biochem. 1995; 64: 653-688Crossref PubMed Scopus (226) Google Scholar)). The relationship of each step to membrane behavior varies among the different isoforms (17.Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 18.Singer A.G. Ghomashchi F. Le Calvez C. Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. Gelb M.H. J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 19.Pan Y.H. Yu B.Z. Singer A.G. Ghomashchi F. Lambeau G. Gelb M.H. Jain M.K. Bahnson B.J. J. Biol. Chem. 2002; 277: 29086-29093Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 20.Beers S.A. Buckland A.G. Giles N. Gelb M.H. Wilton D.C. Biochemistry. 2003; 42: 7326-7338Crossref PubMed Scopus (46) Google Scholar). A prominent example is the degree to which initial adsorption (step 1) depends on the presence of negative charge at the membrane surface (18.Singer A.G. Ghomashchi F. Le Calvez C. Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. Gelb M.H. J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 21.Bezzine S. Bollinger J.G. Singer A.G. Veatch S.L. Keller S.L. Gelb M.H. J. Biol. Chem. 2002; 277: 48523-48534Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 22.Bell J.D. Biltonen R.L. J. Biol. Chem. 1989; 264: 225-230Abstract Full Text PDF PubMed Google Scholar). For instance, an anionic membrane surface appears required for pancreatic and hGIIa sPLA2 (18.Singer A.G. Ghomashchi F. Le Calvez C. Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. Gelb M.H. J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 23.Jain M.K. Yu B.Z. Kozubek A. Biochim. Biophys. Acta. 1989; 980: 23-32Crossref PubMed Scopus (93) Google Scholar). For hGV, the presence of a tryptophan residue at the interfacial binding surface of the enzyme diminishes this requirement and allows some adsorption to a zwitterionic interface (17.Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 24.Han S.K. Yoon E.T. Cho W. Biochem. J. 1998; 331: 353-357Crossref PubMed Scopus (53) Google Scholar). In the case of the hGX enzyme, there appears to be a smaller or perhaps no requirement for an anionic interface (18.Singer A.G. Ghomashchi F. Le Calvez C. Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. Gelb M.H. J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 19.Pan Y.H. Yu B.Z. Singer A.G. Ghomashchi F. Lambeau G. Gelb M.H. Jain M.K. Bahnson B.J. J. Biol. Chem. 2002; 277: 29086-29093Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). For snake venom sPLA2 from Agkistrodon piscivorus piscivorus, either or both steps may be limiting depending on the physical state of the membrane (15.Henshaw J.B. Olsen C.A. Farnbach A.R. Nielson K.H. Bell J.D. Biochemistry. 1998; 37: 10709-10721Crossref PubMed Scopus (43) Google Scholar). This diversity of behaviors among sPLA2 isoforms suggests that they would respond differently to membrane changes induced by apoptosis. For example, migration of phosphatidylserine from the inner to outer surface of the cell membrane during apoptosis is an obvious means by which sPLA2 isoforms sensitive to negative charge might be capacitated to hydrolyze the membrane (25.Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Recent investigations have revealed additional changes such as increased interlipid spacing that may influence other isoforms (9.Bailey R.W. Nguyen T. Robertson L. Gibbons E. Nelson J. Christensen R.E. Bell J.P. Judd A.M. Bell J.D. Biophys. J. 2009; 96: 2709-2718Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). This study is designed to explore sPLA2 species that differ in their membrane requirements and compare their response to bilayer physical properties associated with calcium ionophore and glucocorticoid-initiated apoptosis. Three human isoforms are included (hGIIa, hGV, and hGX). Snake venom (A. p. piscivorus) sPLA2 (AppD49) is used as a standard for internal comparison to previous studies. The monomeric aspartate 9 phospholipase A2 from the venom of A. p. piscivorus was isolated according to the procedure of Maraganore et al. (26.Maraganore J.M. Merutka G. Cho W. Welches W. Kézdy F.J. Heinrikson R.L. J. Biol. Chem. 1984; 259: 13839-13843Abstract Full Text PDF PubMed Google Scholar). The following recombinant human sPLA2 isoforms were prepared as described previously: hGIIA (27.Baker S.F. Othman R. Wilton D.C. Biochemistry. 1998; 37: 13203-13211Crossref PubMed Scopus (86) Google Scholar, 28.Markova M. Koratkar R.A. Silverman K.A. Sollars V.E. MacPhee-Pellini M. Walters R. Palazzo J.P. Buchberg A.M. Siracusa L.D. Farber S.A. Oncogene. 2005; 24: 6450-6458Crossref PubMed Scopus (18) Google Scholar), hGV (29.Cho W. Han S.K. Lee B.I. Snitko Y. Dua R. Methods Mol. Biol. 1999; 109: 31-38PubMed Google Scholar), and hGX (19.Pan Y.H. Yu B.Z. Singer A.G. Ghomashchi F. Lambeau G. Gelb M.H. Jain M.K. Bahnson B.J. J. Biol. Chem. 2002; 277: 29086-29093Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The hGV enzyme was generously provided by Dr. Wonhwa Cho (University of Illinois, Chicago). Dexamethasone and ionomycin were dissolved in dimethyl sulfoxide (DMSO). Acrylodan-labeled fatty acid-binding protein (ADIFAB), propidium iodide, cell culture medium, and serum were acquired from Invitrogen. S49 mouse lymphoma cells were grown as a suspension culture at 37 °C in humidified air containing 10% CO2 as explained (30.Wilson H.A. Huang W. Waldrip J.B. Judd A.M. Vernon L.P. Bell J.D. Biochim. Biophys. Acta. 1997; 1349: 142-156Crossref PubMed Scopus (25) Google Scholar). Samples treated with dexamethasone received the drug (100 nm final) 6–48 h before harvesting. Control samples received a corresponding volume of DMSO (0.02% v/v). For hydrolysis assays, cells were harvested by gentle centrifugation, washed, and suspended (0.4–3.5 × 106 cells/ml) in a balanced salt medium (MBSS: NaCl = 134 mm, KCl = 6.2 mm, CaCl2 = 1.6 mm, MgCl2 = 1.2 mm, Hepes = 18.0 mm, and glucose = 13.6 mm, pH 7.4, 37 °C). Samples were transferred to quartz fluorometer sample cells and equilibrated for at least 5 min in a spectrofluorometer (Fluoromax 3, Horiba Jobin Yvon, Edison, NJ). Temperature and sample homogeneity were maintained using a water-jacketed sample chamber equipped with magnetic stirring and attached to a circulating water bath. All experiments were performed at 37 °C. The acrylodan-derivatized fatty acid-binding protein, ADIFAB, was used to assay the release of fatty acids from cell membranes in real time. Data were acquired from cell samples 100 s (excitation = 390 nm, emission = 432 and 505 nm, bandpass = 4 nm) before adding ADIFAB (65 nm final) to assay background intensity. After the addition of ADIFAB and stabilization of the fluorescence intensity (about 300–500 s), one of the four sPLA2 isoforms was added (0.07–70 nm final), and the time course was continued for an additional 800–2000 s. Fatty acid release was estimated by transforming the raw intensities to generalized polarization values (GP) and then fitting to an arbitrary function by nonlinear regression as described (31.Harris F.M. Smith S.K. Bell J.D. J. Biol. Chem. 2001; 276: 22722-22731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). ADIFABGP=(l505−l432)(l505+l432)(Eq. 1) I505 and I432 are the fluorescence emission intensities at 505 and 432 nm. In experiments involving calcium ionophore, ionomycin (300 nm final) or the equivalent diluent (0.25% DMSO) was included in samples with ADIFAB for at least 300 s before adding sPLA2. The intensity of propidium iodide fluorescence was used to quantify the fraction of cells susceptible to hydrolysis by sPLA2 in samples treated with dexamethasone or equivalent DMSO. Cells were harvested, incubated, and mixed with sPLA2 isoforms as explained above for ADIFAB experiments with differences; propidium iodide (37 μm final) was included instead of ADIFAB, fluorescence intensity was assayed at 617 nm (excitation = 536 nm), and ionomycin was added at the end of the time course to render all the cells hydrolyzable and, thus, provide a maximum signal for internal comparison (9.Bailey R.W. Nguyen T. Robertson L. Gibbons E. Nelson J. Christensen R.E. Bell J.P. Judd A.M. Bell J.D. Biophys. J. 2009; 96: 2709-2718Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Data were analyzed by nonlinear regression and by quantifying the various subpopulations as described previously (9.Bailey R.W. Nguyen T. Robertson L. Gibbons E. Nelson J. Christensen R.E. Bell J.P. Judd A.M. Bell J.D. Biophys. J. 2009; 96: 2709-2718Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The subpopulation of interest for this study was that in which cells were still alive (i.e. excluded propidium iodide) but susceptible to sPLA2 (i.e. incorporated the dye upon the addition of sPLA2). This subpopulation was quantified by calculating the difference in propidium iodide fluorescence intensity before and after the addition of sPLA2 and normalizing to the maximum fluorescence change observed in samples treated with both AppD49 sPLA2 and ionomycin. Data were corrected for variations in cell number using direct cell counts (by light microscopy) and light scatter intensity. Because the emission spectra of ADIFAB and propidium iodide do not overlap, the fluorescence of the two probes was assayed simultaneously for some experiments (Fig. 1) as described previously (30.Wilson H.A. Huang W. Waldrip J.B. Judd A.M. Vernon L.P. Bell J.D. Biochim. Biophys. Acta. 1997; 1349: 142-156Crossref PubMed Scopus (25) Google Scholar). These experiments were done in parallel with spectrofluorometric measurements of hydrolysis and propidium iodide uptake. Cells were washed and suspended in MBSS. After washing, aliquots of the control and treatment samples were transferred to flow cytometry sample tubes and incubated for 5 min with propidium iodide (10 μm final). Cell subpopulations were then identified based on the level of fluorescence intensity using a BD FACSCanto flow cytometer (BD Biosciences) with excitation at 488 nm, and emission was detected in the range of 564–606 nm. Fig. 1A displays a time course of hydrolysis of a sample of S49 cells by extracellular AppD49 sPLA2. As is typical for healthy untreated samples, a small amount of fatty acid (and lysophospholipid) was produced upon the addition of sPLA2 followed by a gradual return to base line as the reaction ended, and the products were salvaged by reacylation (13.Wilson H.A. Waldrip J.B. Nielson K.H. Judd A.M. Han S.K. Cho W. Sims P.J. Bell J.D. J. Biol. Chem. 1999; 274: 11494-11504Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This transient burst of hydrolysis was accompanied by a small increase in the permeability of the cells to the fluorescent dye propidium iodide (Fig. 1B). Normally, healthy cells exclude the dye, but when their membranes are damaged by hydrolysis, the dye enters the cells and binds to DNA and emits with higher intensity (10.Nielson K.H. Olsen C.A. Allred D.V. O'Neill K.L. Burton G.F. Bell J.D. Biochim. Biophys. Acta. 2000; 1484: 163-174Crossref PubMed Scopus (24) Google Scholar). This small rise in propidium iodide fluorescence, thus, represents cells that are initially alive (impermeable) but are then killed by the action of the phospholipase (labeled Alive & Susceptible in the Fig. 1 inset). Although the increased intensity in Fig. 1B was very small, it was consistent with previous studies comparing healthy and apoptotic cells (10.Nielson K.H. Olsen C.A. Allred D.V. O'Neill K.L. Burton G.F. Bell J.D. Biochim. Biophys. Acta. 2000; 1484: 163-174Crossref PubMed Scopus (24) Google Scholar). In contrast, when ionomycin was subsequently introduced into the sample, more extensive hydrolysis was observed, as indicated by a large elevation of free fatty acid (Fig. 1A) and propidium iodide emission (Fig. 1B). The enhanced enzymatic activity and resulting membrane damage upon the addition of ionomycin represented a hydrolytic attack of 100% of the cells as the level of propidium iodide fluorescence could not be further increased by subsequent incubation of the sample with a detergent (Triton X-100, 0.25% v/v). Interpretation of the experiments in this study requires that we determine whether the data in Fig. 1A represent uniform levels of hydrolysis among all the cells in the sample or extensive sensitivity of a small subpopulation of vulnerable cells. This question is of particular concern for experiments involving sPLA2 because the products of hydrolysis from one cell may be able to diffuse to another cell and induce hydrolysis as has been observed with artificial membranes (32.Bell J.D. Brown S.D. Baker B.L. Biochim. Biophys. Acta. 1992; 1127: 208-220Crossref PubMed Scopus (10) Google Scholar). The matter was addressed by analysis of hydrolysis time courses in the context of flow cytometry data gathered on the same samples as explained below (Figs. 1, C and D). Previously, the transient burst of activity in control samples (Fig. 1A) was assumed to represent minor hydrolysis of all cells. To account for the complete cessation of hydrolysis even though substrate was restored by reacylation, a model was created in which cells become refractory after initial exposure to sPLA2 (13.Wilson H.A. Waldrip J.B. Nielson K.H. Judd A.M. Han S.K. Cho W. Sims P.J. Bell J.D. J. Biol. Chem. 1999; 274: 11494-11504Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). However, an alternative model in which a minority of the cells is susceptible to the enzyme and the remainder is resistant can also account quantitatively for the result if two assumptions are made. First, hydrolysis ceases because the membranes of vulnerable cells have been consumed. Second, uptake and reacylation of reaction products is accomplished by the remaining healthy cells in the sample. To distinguish these possibilities, we used flow cytometry to separate healthy cell samples into subpopulations based on propidium iodide permeability (Fig. 1C). Data were then compared with and without sPLA2 treatment to see whether all the cells were uniformly affected by the enzyme or whether certain subpopulations were preferentially altered. As shown in Fig. 1C, three subpopulations were identified as peaks in the flow cytometry histogram. Peak 1 represented cells that did not stain with propidium iodide. Peak 2 represented cells that displayed very low permeability to propidium iodide, an observation described for thymocytes early during apoptosis and necrosis (33.Lyons A.B. Samuel K. Sanderson A. Maddy A.H. Cytometry. 1992; 13: 809-821Crossref PubMed Scopus (99) Google Scholar, 34.Vitale M. Zamai L. Mazzotti G. Cataldi A. Falcieri E. Histochemistry. 1993; 100: 223-229Crossref PubMed Scopus (95) Google Scholar). Although the fluorescence intensity of these cells was 10 times that of background (Peak 1), it represented only 1% that observed for cells that were fully permeable to the dye (Peak 3). The complete permeability of the subpopulation shown in Peak 3 was confirmed by comparison to samples treated with the detergent Triton X-100 (0.1% v/v). Traditionally, cells arriving spontaneously in Peak 3 would be considered necrotic (34.Vitale M. Zamai L. Mazzotti G. Cataldi A. Falcieri E. Histochemistry. 1993; 100: 223-229Crossref PubMed Scopus (95) Google Scholar). Previous studies have suggested that necrotic cells are fully susceptible to hydrolysis by sPLA2 (8.Bailey R.W. Olson E.D. Vu M.P. Brueseke T.J. Robertson L. Christensen R.E. Parker K.H. Judd A.M. Bell J.D. Biophys. J. 2007; 93: 2350-2362Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 13.Wilson H.A. Waldrip J.B. Nielson K.H. Judd A.M. Han S.K. Cho W. Sims P.J. Bell J.D. J. Biol. Chem. 1999; 274: 11494-11504Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 30.Wilson H.A. Huang W. Waldrip J.B. Judd A.M. Vernon L.P. Bell J.D. Biochim. Biophys. Acta. 1997; 1349: 142-156Crossref PubMed Scopus (25) Google Scholar). This interpretation was confirmed indirectly using samples that contained varied proportions of necrotic cells (6–81%). Aliquots of these samples were analyzed by flow cytometry in parallel with measurements of hydrolysis using ADIFAB. A very strong correlation was observed with respect to the amount of hydrolysis and the area of Peak 3 in the flow cytometry histogram (r2 = 0.93, p < 0.0001, n = 9). Moreover, the slope of the regression line was 0.91 ± 0.09, suggesting that the majority of the hydrolysis observed could be accounted for by the fully-permeable, necrotic cells of Peak 3. We were able to assess the hydrolytic susceptibility of the cells in Peak 2 more directly because these cells were only modestly permeable to propidium iodide, and hydrolysis would, therefore, have a distinct measurable effect by making the subpopulation more permeable. Accordingly, the area defined by this part of the histogram was compared before and after treatment with sPLA2. As shown by the dashed curve in Fig. 1C, Peak 2 was reduced dramatically after exposure to sPLA2 (p = 0.002, n = 19), presumably because the cells had become permeabilized by the enzyme and, thus, shifted into Peak 3. It is likely that the shallow slope of the time course in Fig. 1B represents the low permeability of the cells designated by Peak 2. If this is the case, the slope should be reduced after treatment with sPLA2 depletes the subpopulation. In fact, the slope of the time profile was reduced by 35% (p = 0.02, n = 6) upon the addition of sPLA2, again suggesting that the small burst of propidium iodide fluorescence seen in the inset of Fig. 1B represents hydrolytic attack of this tiny subpopulation. The large population of healthy impermeable cells in Peak 1 was also analyzed as described for Peak 2. In this case, the subpopulation was mostly unaffected by sPLA2; the area of Peak 1 was identical regardless of the presence of the phospholipase (Fig. 1C, p = 0.21, n = 19). Nevertheless, the mode of the peak was shifted slightly (7% increase in propidium iodide intensity compared with the 1000-fold increase when cells become fully permeable) but reproducibly (p = 0.006, n = 19). This result suggests that the enzyme probably does hydrolyze lipids of healthy cells but that the level is extremely small compared with the attack of the Peak 2 and Peak 3 subpopulations. To determine whether hydrolysis of these two apparently vulnerable subpopulations could account quantitatively for the transient fatty acid release observed from ADIFAB data, we added the area of Peaks 2 and 3 and compared that sum to the size of the hydrolysis data. As shown in Fig. 1D, the size of these two subpopulations and the amount of hydrolysis observed in aliquots from the same samples were indistinguishable (p = 0.74, n = 6). Even though these results are based on correlation, they demonstrated that a simple model can account quantitatively for the observation of transient hydrolysis rather than having to invoke the complex ideas proposed previously (13.Wilson H.A. Waldrip J.B. Nielson K.H. Judd A.M. Han S.K. Cho W. Sims P.J. Bell J.D. J. Biol. Chem. 1999; 274: 11494-11504Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Therefore, we concluded that the hydrolysis of healthy cell samples by sPLA2, as observed with ADIFAB fluorescence, reflects the action of the enzyme on small vulnerable subpopulations without adverse impact on the remaining cells. Treatment of S49 cells for 18 h with dexamethasone produced a level of hydrolysis by AppD49 sPLA2 intermediate between that observed in control versus ionomycin-treated samples (Fig. 2). As in Fig. 1A, subsequent addition of ionomycin induced hydrolysis of the remaining sample. Hence, as in the control samples, hydrolysis in apoptotic cells appeared confined to a sensitive subpopulation. Treatments, then, that enhan" @default.
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