FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1994792016> ?p ?o ?g. }

• W1994792016 endingPage "18129" @default.
• W1994792016 startingPage "18122" @default.
• W1994792016 abstract "Aerolysin is a pore-forming toxin that plays a key role in the pathogenesis of Aeromonas hydrophilainfections. In this study, we have analyzed the effect of aerolysin on human granulocytes (HL-60 cells). Proaerolysin could bind to these cells, was processed into active aerolysin, and led to membrane depolarization, indicating that granulocytes are potential targets for this toxin. Fura-2 measurements were used to analyze the effect of aerolysin on cytosolic [Ca2+] homeostasis. As expected for a pore-forming toxin, aerolysin addition led to Ca2+influx across the plasma membrane. In addition, the toxin triggered Ca2+ release from agonist and thapsigargin-sensitive intracellular Ca2+ stores. This Ca2+ release was independent of the aerolysin-induced Ca2+ influx and occurred in two kinetically distinct phases: an initial rapid and transient phase and a second, more sustained, phase. The first, but not the second phase was sensitive to pertussis toxin. Activation of pertussis toxin-sensitive G-proteins appeared to be a consequence of pore formation, rather than receptor activation through aerolysin-binding, as it: (i) was not observed with a binding competent, insertion-incompetent aerolysin mutant, (ii) had a marked lag time, and (iii) was also observed in response to other bacterial pore-forming toxins (staphylococcal α-toxin, streptolysin O) which are thought to bind to different receptors. G-protein activation through pore-forming toxins stimulated cellular functions, as evidenced by pertussis toxin-sensitive chemotaxis. Our results demonstrate that granulocytes are potential target cells for aerolysin and that in these cells, Ca2+ signaling in response to a pore-forming toxin involves G-protein-dependent cell activation and Ca2+ release from intracellular stores. Aerolysin is a pore-forming toxin that plays a key role in the pathogenesis of Aeromonas hydrophilainfections. In this study, we have analyzed the effect of aerolysin on human granulocytes (HL-60 cells). Proaerolysin could bind to these cells, was processed into active aerolysin, and led to membrane depolarization, indicating that granulocytes are potential targets for this toxin. Fura-2 measurements were used to analyze the effect of aerolysin on cytosolic [Ca2+] homeostasis. As expected for a pore-forming toxin, aerolysin addition led to Ca2+influx across the plasma membrane. In addition, the toxin triggered Ca2+ release from agonist and thapsigargin-sensitive intracellular Ca2+ stores. This Ca2+ release was independent of the aerolysin-induced Ca2+ influx and occurred in two kinetically distinct phases: an initial rapid and transient phase and a second, more sustained, phase. The first, but not the second phase was sensitive to pertussis toxin. Activation of pertussis toxin-sensitive G-proteins appeared to be a consequence of pore formation, rather than receptor activation through aerolysin-binding, as it: (i) was not observed with a binding competent, insertion-incompetent aerolysin mutant, (ii) had a marked lag time, and (iii) was also observed in response to other bacterial pore-forming toxins (staphylococcal α-toxin, streptolysin O) which are thought to bind to different receptors. G-protein activation through pore-forming toxins stimulated cellular functions, as evidenced by pertussis toxin-sensitive chemotaxis. Our results demonstrate that granulocytes are potential target cells for aerolysin and that in these cells, Ca2+ signaling in response to a pore-forming toxin involves G-protein-dependent cell activation and Ca2+ release from intracellular stores. Aerolysin is a pore-forming toxin secreted by the human pathogenAeromonas hydrophila and has been shown to be an important virulence factor produced by this bacterium (1Donta S.T. Haddow A.P. Infect. Immun. 1978; 21: 989-993Crossref PubMed Google Scholar, 2Daily O.P. Joseph S.W. Coolbaugh J.C. Walker R.I. Merrell B.R. Rollins D.M. Seidler R.J. Colwell R.R. Lissner C.R. J. Clin. Microbiol. 1981; 13: 769-777Crossref PubMed Google Scholar, 3Kaper J.B. Lockman H. Colwell R.R. J. Appl. Bacteriol. 1981; 50: 359-377Crossref PubMed Scopus (160) Google Scholar, 4Janda J.M. Bottone D.J. Sinner C.V. Calcaterra D. J. Clin. Microbiol. 1984; 17: 588-591Crossref Google Scholar, 5Chakraborty T. Hihle B. Berghauer H. Goebel W. Infect. Immun. 1987; 55: 2274-2280Crossref PubMed Google Scholar). A. hydrophila has been implicated in a variety of diseases ranging from gastroenteritis to deep wound infection and septicemia. The importance of aerolysin in the pathogenicity of the bacterium is best illustrated by the fact that immunization against the toxin leads to protection toward the bacterium. The toxin is secreted by A. hydrophila as a dimeric inactive precursor (6Howard S.P. Buckley J.T. Biochemistry. 1982; 21: 1662-1667Crossref PubMed Scopus (106) Google Scholar, 7van der Goot F. Ausio J. Wong K. Pattus F. Buckley J. J. Biol. Chem. 1993; 268: 18272-18279Abstract Full Text PDF PubMed Google Scholar) which can be activated by proteolytic cleavage of a C-terminal peptide (8Howard S.P. Buckley J.T. J. Bacteriol. 1985; 163: 336-340Crossref PubMed Google Scholar, 9van der Goot F.G. Lakey J.H. Pattus F. Kay C.M. Sorokine O. Van Dorsselaer A. Buckley T. Biochemistry. 1992; 31: 8566-8570Crossref PubMed Scopus (73) Google Scholar). The toxin as well as the protoxin interact with the target cell by binding to specific receptors (10Gruber H.J. Wilmsen H.U. Cowell S. Schindler H. Buckley J.T. Mol. Microbiol. 1994; 14: 1093-1101Crossref PubMed Scopus (25) Google Scholar, 11Nelson K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12Cowell S. Aschauer W. Gruber H.J. Nelson K.L. Buckley J.T. Mol. Microbiol. 1997; 25: 343-350Crossref PubMed Scopus (52) Google Scholar, 13Diep D.B. Nelson K.L. Raja S.M. Pleshak E.N. Buckley J.T. J. Biol. Chem. 1998; 273: 2355-2360Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 14Abrami L. Fivaz M. Buckley J.T. Parton R.G. van der Goot F.G. J. Cell. Biol. 1998; 140: 525-540Crossref PubMed Scopus (195) Google Scholar). At present all identified receptors were found to be GPI 1The abbreviations used are: GPI, glycosylphosphatidylinositol; PAGE, polyacrylamide gel electrophoresis; PLC, phospholipase C; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Me2SO, dimethyl sulfoxide; ER, endoplasmic reticulum; fMLP, fMet-Leu-Phe; PBFI, K+-binding benzofuran isophthalate. 1The abbreviations used are: GPI, glycosylphosphatidylinositol; PAGE, polyacrylamide gel electrophoresis; PLC, phospholipase C; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Me2SO, dimethyl sulfoxide; ER, endoplasmic reticulum; fMLP, fMet-Leu-Phe; PBFI, K+-binding benzofuran isophthalate. anchored. However, different receptors were found on different cells types and a given cell type was found to have more then one receptor. For example, aerolysin was shown to bind to Thy-1 as well as other GPI-anchored proteins on T-lymphocytes, to Thy-1 and contactin in mouse brain (11Nelson K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar,13Diep D.B. Nelson K.L. Raja S.M. Pleshak E.N. Buckley J.T. J. Biol. Chem. 1998; 273: 2355-2360Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), to VSG from Trypanosomes (13Diep D.B. Nelson K.L. Raja S.M. Pleshak E.N. Buckley J.T. J. Biol. Chem. 1998; 273: 2355-2360Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), to an 47-kDa receptor on rat erythrocytes (12Cowell S. Aschauer W. Gruber H.J. Nelson K.L. Buckley J.T. Mol. Microbiol. 1997; 25: 343-350Crossref PubMed Scopus (52) Google Scholar) and to mainly an 80-kDa receptor on baby hamster kidney cells (14Abrami L. Fivaz M. Buckley J.T. Parton R.G. van der Goot F.G. J. Cell. Biol. 1998; 140: 525-540Crossref PubMed Scopus (195) Google Scholar). Binding was shown to be determined both by the protein moiety and the olisaccharides of the anchor (13Diep D.B. Nelson K.L. Raja S.M. Pleshak E.N. Buckley J.T. J. Biol. Chem. 1998; 273: 2355-2360Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Binding to the cell surface presumably leads to a local increase in toxin concentration thereby enabling aerolysin to polymerize into a heptameric complex that inserts into the membrane and forms a water-filled channel (6Howard S.P. Buckley J.T. Biochemistry. 1982; 21: 1662-1667Crossref PubMed Scopus (106) Google Scholar, 15Garland W.J. Buckley J.T. Infect. Immun. 1988; 56: 1249-1253Crossref PubMed Google Scholar, 16Wilmsen H.U. Pattus F. Buckley J.T. J. Membr. Biol. 1990; 115: 71-81Crossref PubMed Scopus (88) Google Scholar, 17Wilmsen H.U. Leonard K.R. Tichelaar W. Buckley J.T. Pattus F. EMBO J. 1992; 11: 2457-2463Crossref PubMed Scopus (101) Google Scholar, 18Moniatte M. van der Goot F.G. Buckley J.T. Pattus F. Van Dorsselaer A. FEBS Lett. 1996; 384: 269-272Crossref PubMed Scopus (95) Google Scholar). Cells such as erythrocytes, that are unable to cope with such membrane damage, undergo osmotic lysis. In nucleated mammalian cells, the mechanisms leading to cell death appear to be more complex. We have indeed recently found that subnanomolar doses of aerolysin do not induce lysis of baby hamster kidney cells (14Abrami L. Fivaz M. Buckley J.T. Parton R.G. van der Goot F.G. J. Cell. Biol. 1998; 140: 525-540Crossref PubMed Scopus (195) Google Scholar). Permeabilization but not disruption of the plasma membrane was observed followed by selective vacuolation of the endoplasmic reticulum (14Abrami L. Fivaz M. Buckley J.T. Parton R.G. van der Goot F.G. J. Cell. Biol. 1998; 140: 525-540Crossref PubMed Scopus (195) Google Scholar). Only several hours later could a loss of plasma membrane integrity be observed. It is at present not clear whether the pores formed by the toxin at the plasma membrane are the sole cause of the observed effects. These findings, however, do suggest that aerolysin may trigger a cascade of events from the plasma membrane. In this study, we have analyzed the effects of aerolysin on human granulocytes. We show that, in addition to formation of pores in the plasma membrane, aerolysin triggered, through activation of a pertussis toxin-sensitive G-protein, chemotaxis, and release of Ca2+from intracellular stores. Cell culture media were obtained from Life Technologies, Inc. (Paisley, Scotland), U73122 from Calbiochem (La Jolla, CA), and DiSC3(5), PBFI, and fura-2/AM from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma or Fluka. The “Ca2+-free medium” contained: 143 mm NaCl, 6 mm KCl, 1 mmMgSO4, 5.6 mm glucose (0.1%), 20 mm HEPES pH 7.4, and 0.1 mm EGTA. The “Ca2+ medium” consisted of Ca2+-free medium supplemented with 1 mm CaCl2. HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (50 units/ml), streptomycin (50 μg/ml), andl-glutamine (2 mm) at 37 °C in a humidified atmosphere of 5% CO2, 95% air. Granulocytic differentiation was initiated by addition of dimethyl sulfoxide (Me2SO) (final concentration 1.3% for 3 days, then 0.65% for 1 or 2 days). Wild type and variant proaerolysins were purified as described previously (19Buckley J.T. Biochem. Cell Biol. 1990; 68: 221-224Crossref PubMed Scopus (54) Google Scholar). Concentrations were determined by measuring the optical density (O.D.) at 280 nm, considering that a 1 mg/ml sample has an O.D. of 2.5 (20van der Goot F.G. Hardie K.R. Parker M.W. Buckley J.T. J. Biol. Chem. 1994; 269: 30496-30501Abstract Full Text PDF PubMed Google Scholar). Proaerolysin was labeled with 125I using IODO-GEN reagent (Pierce) according to the manufacturers recommendations.125I-Proaerolysin was separated from the free iodine by gel filtration on a PD10-G25 column (Pharmacia, Sweden) equilibrated with phosphate-buffered saline. We consistently obtained a specific activity of about 2 × 106 cpm/μg of proaerolysin.125I-Proaerolysin ran as a single band on an SDS gel. Aerolysin was obtained by treating proaerolysin with trypsin at a protein to enzyme ratio of 1/20 (mol:mol) for 10 min at room temperature. After which a 10-fold excess of trypsin inhibitor was added. HL-60 granulocytes at a concentration of 2 × 106 cells/ml in Ca2+medium were incubated with 125I-proaerolysin for 25 min at 4 °C, spun down in a table top cell centrifuge for 8 min at 1600 rpm, resuspended in the same volume of buffer. The last washing step was performed twice. In competition experiments,125I-proaerolysin and the unlabeled wild type or mutant toxin were added to the cells simultaneously. For SDS-PAGE analysis, cells were briefly sonicated with a tip sonicator in sample buffer. SDS-PAGE was performed as described by Laemmli (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). HL-60 granulocytes were washed once and resuspended in buffer containing 20 mmHEPES pH 7.4, 143 mm NaCl, 5 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 5.6 mm glucose, to a final density of 3 × 106 cells/ml. DiS-C3(5) (100 μmin Me2SO) was added to a final concentration of 200 nm. Membrane incorporation of the dye was monitored spectrofluorimetrically using a Photon Technology International fluorometer equipped with a thermostated cuvette holder (excitation 625 nm; emission 670 nm; 10 nm slits). After reaching a steady state fluorescence, the toxin was added. Maximal depolarization was obtained at the end of each experiment by adding pre-mixed valinomycin and nigericin to final concentrations of 2 and 5 μm, respectively (22Kasner S.E. Ganz M.B. Am. J. Physiol. 1992; 262: F462-F467Crossref PubMed Google Scholar). Single fluorescent traces were expressed as the ratio I(t)/I max,i.e. fluorescence intensity at a given time over maximal fluorescence intensity. HL-60 granulocytes were washed once and resuspended in loading medium, containing 20 mm HEPES pH 7.4, 5.6 mm glucose, 143 mm NaCl, 6 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 0.5% bovine serum albumin, and 0.25 mm sulfinpyrazone, to a final density of 20 × 106 cells/ml (22Kasner S.E. Ganz M.B. Am. J. Physiol. 1992; 262: F462-F467Crossref PubMed Google Scholar). Cells were incubated with the cell-permeant form of the K+-binding benzofuran isophtalate dye PBFI-AM (stock solution in Me2SO in presence of pluronic acid F-127, final concentrations of 5 μm PBFI and 0.02% F-127) for 30 min at 37 °C, followed by 30 min at room temperature, washed once and resuspended in the same buffer, in the absence of bovine serum albumin, to a final density of 2 × 106 cells/ml. Fluorescence measurements were performed using a Photon Technology International fluorometer. The excitation and emission wavelengths were 343 and 460 nm, respectively (37 °C). Variations of intracellular K+contents were expressed as a fraction of PBFI maximal intensity. HL-60 granulocytes were washed once and resuspended in 20 mm HEPES pH 7.4, 5.6 mm glucose, 143 mm NaCl, 6 mm KCl, 1 mmMgSO4, 1 mm CaCl2, to a final density of 20 × 106 cells/ml. Ethidium homodimer-1 (stock solution 2 mm in Me2SO/water, 1:4) or ethidium bromide (stock solution 10 mg/ml in water) were added to a final concentration of 6 nm and 100 μm, respectively. Aerolysin-induced ethidium homodimer-1 or ethidium bromide entry was monitored by measuring the increase of fluorescence intensity at 600 nm, upon excitation at 500 or 340 nm, respectively. Single fluorescent traces were normalized to maximal fluorescence obtained by the addition of 1% Triton X-100. [Ca2+]c was measured with the fluorescent Ca2+ indicator fura-2. Cells (2 × 107/ml) suspended in Ca2+ medium containing 0.1% bovine serum albumin were loaded for 45 min at 37 °C with 2 μm fura-2/AM, then diluted to 107/ml and kept on ice. Just before use, a sample of loaded cells (2 × 106/ml) was centrifuged and resuspended in the desired medium. Fluorescence measurements were performed on a Perkin-Elmer fluorometer (LS3, Perkin-Elmer), thermostated at 37 °C. Excitation and emission wavelengths were 340 and 505 nm, respectively. Calibration was performed for each cuvette by sequential addition of 2 mm Ca2+ (for Ca2+-free medium), 1 μm ionomycin to measure Ca2+ saturated fura-2 (F max), followed by 24 mm EGTA, 75 mm Tris, pH 9.3, and 0.1% Triton X-100 to measure Ca2+ free fura-2 (F min). A relatively small leakage of fura-2 occurred in cells exposed to aerolysin (see “Results”). Results are shown as relative fura-2 fluorescence, normalized with respect to the maximal fluorescence (=100%) and minimal fluorescence (=0%) values obtained through the calibration procedure. At an excitation wavelength of 360 nm, fura-2 fluorescence is Ca2+ independent, the fluorescence of the probe is, however, quenched by several divalent cations. In this study, we used this feature of the probe to study entry of Mn2+ and Ni2+ in response to aerolysin independently from changes in [Ca2+]c. Cell-associated fluorescence before addition of the respective divalent cation was defined as 100% fluorescence. For the quantitation of the Mn2+ influx at different times after aerolysin addition, we proceeded as described previously (23Demaurex N. Monod A. Lew D. Krause K.-H. Biochem. J. 1994; 297: 595-601Crossref PubMed Scopus (81) Google Scholar). Briefly, the percentage of fluorescence quenching that occurred within 1 min after Mn2+ addition was determined. The relatively small fraction of the fluorescence quenching that was due to the presence of extracellular fura (see below) was subtracted. The emission wavelength was 505 nm. To determine the amount of extracellular fura-2, we exposed fura-2-loaded cells for various times to aerolysin, followed by the addition of 25 μm Mn2+ and, 1 min later, 100 μm of the heavy metal chelator diethylenetriaminepentaacetic acid. Under these conditions, the fraction of the Mn2+-induced fluorescence quenching that is immediately reversible after addition diethylenetriaminepentaacetic acid is a direct measure of extracellular fura-2 (23Demaurex N. Monod A. Lew D. Krause K.-H. Biochem. J. 1994; 297: 595-601Crossref PubMed Scopus (81) Google Scholar). For the chemotaxis assay, a Transwell® chemotaxis chamber (6.5 mm diameter, 3 μm pore size, Corning Costar Corp., Cambridge, MA) was used. In this system, the cell reservoir (=upper chamber) is separated from the target chamber (=lower chamber) by a microporous membrane. The cell reservoir contained 106cells in 100 μl of Ca2+ buffer with 0.1% bovine serum albumin. The target chamber contained the indicated concentration of chemoattractant or the appropriate solvent control in 500 μl of Ca2+ buffer with 0.1% bovine serum albumin. Chemotaxis was allowed to occur over a period of 90 min in an CO2 (5%) and temperature (37 °C)-controlled incubator. Cells in the target chamber were counted. Results are expressed as cells recovered in the target chamber (% of cells that were initially added in the cell reservoir). As a first step in the characterization of the interaction of proaerolysin with myeloid cells, we have investigated whether proaerolysin was able to bind to HL-60 promyelocytes and HL-60 granulocytes. Cells were incubated with 1 nm proaerolysin at 4 °C for 10 min, washed, sedimented, and analyzed by SDS-PAGE followed by Western blot analysis for the presence of the toxin. Both wild type proaerolysin as well as a double cysteine mutant, G202C/I445C (see below), were able to bind to both types of HL-60 cells (not shown). To investigate whether binding of proaerolysin was specific, we have analyzed whether radiolabeled and unlabeled proaerolysin could compete for binding to HL-60 cells. Binding of radiolabeled proaerolysin (4 nm) to both promyelocytic and granulocytic HL-60 could be inhibited by more than 80% by the presence of a 100-fold excess of unlabeled toxin (0.4 μm), indicating the presence of a limited number of binding sites. Binding of radiolabeled wild type toxin could also be inhibited, to the same extend, by unlabeled G202C/I445C mutant toxin, indicating that both forms of the toxin bind to the same sites. These observations suggest that aerolysin binds to a limited number of sites on HL-60 cells. Using a previously described proaerolysin overlay assay (14Abrami L. Fivaz M. Buckley J.T. Parton R.G. van der Goot F.G. J. Cell. Biol. 1998; 140: 525-540Crossref PubMed Scopus (195) Google Scholar), we could identify 4 proaerolysin-binding proteins (not shown). Binding to these proteins could be inhibited by 70% by treating the cells with the phosphatidylinositol-specific phospholipase C indicating that these putative receptors were GPI anchored (not shown). These four proteins remain to be identified. We could, however, exclude that binding occurred via Thy-1, which was shown to be a receptor for aerolysin on T-lymphocytes (11Nelson K.L. Raja S.M. Buckley J.T. J. Biol. Chem. 1997; 272: 12170-12174Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), since HL-60 do not express this protein to any significant extent (24Schlossman S.F. Boumsel L. Gilks W. Harlan J.M. Kishimoto T. Morimoto C. Ritz J. Shaw S. Silverstein R. Springer T. Tedder T.F. Todd R.F. Leukcocyte Typing V. White Cell Differentiation Antigens. Oxford University Press, Oxford1995: 154Google Scholar). The presence of multiple receptors on HL-60 granulocytes is reminiscent of what was observed in rat brain, were at least two receptors were found, Thy-1 and contactin (13Diep D.B. Nelson K.L. Raja S.M. Pleshak E.N. Buckley J.T. J. Biol. Chem. 1998; 273: 2355-2360Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and on baby hamster kidney cells were three putative GPI-anchored receptors were seen of, respectively, 140, 80, and 30 kDa, the 80-kDa protein being the major proaerolysin-binding protein (14Abrami L. Fivaz M. Buckley J.T. Parton R.G. van der Goot F.G. J. Cell. Biol. 1998; 140: 525-540Crossref PubMed Scopus (195) Google Scholar). To investigate whether HL-60 granulocytes are sensitive to aerolysin, we have analyzed the effect of the toxin on membrane potential using the fluorescent probe DiSC3(5) which has been widely used for this purpose (25Papini E. Sandona D. Rappuoli R. Montecucco C. EMBO J. 1988; 7: 3353-3359Crossref PubMed Scopus (68) Google Scholar). As shown in Fig. 1 A, proaerolysin led to depolarization of the granulocytes with kinetics that were dose-dependent. As suspected, a marked increase in the rate of depolarization was observed when activating the protoxin prior to the addition to the cells (Fig. 1 B). As shown in Fig. 1 C, depolarization was in part due to the efflux of K+. As a control, we tested the hemolytically inactive mutant of aerolysin, G202C/I445C. This mutant contains two engineered cystein residues that form a disulfide bridge between the propeptide and the mature toxin (20van der Goot F.G. Hardie K.R. Parker M.W. Buckley J.T. J. Biol. Chem. 1994; 269: 30496-30501Abstract Full Text PDF PubMed Google Scholar). Even after trypsin activation, this mutant is unable to lyse erythrocytes presumably because it cannot oligomerize. Also G202C/I445C did not induce K+ efflux from HL-60 granulocytes. Surprisingly, depolarization of HL-60 granulocytes as well as K+ efflux followed two-step kinetics for reasons that remain to be established. In contrast, kinetics of membrane depolarization induced by the thiol-activated toxin streptolysin O were monophasic (not shown). The observed K+ efflux and membrane depolarization were not due to lysis of the cells as illustrated by the fact that most cells still exclude ethidium homodimer-1 after 10 min (Fig. 1 D). Faster kinetics of entry were observed with the smaller dye ethidium bromide indicating that a sieving mechanism was taking place. This observation suggests that the dye enters the cells through the aerolysin pore and not through a breach in the plasma membrane since in the latter case no discrimination in size would be expected. We can therefore conclude that aerolysin led to selective permeabilization of the plasma membrane and not to cell lysis within the time frame of the present experiments. Aerolysin was also able to induce membrane depolarization and K+ efflux in HL-60 promyelocytes, although the kinetics were dramatically slower then those observed for granulocytes (not shown). Since membrane depolarization could be observed not only when treating the cells with aerolysin but also with proaerolysin, albeit at far slower rate, we investigated whether HL-60 granulocytes expressed proteases able to process the protoxin. As shown in Fig. 2, although the protoxin added to the cells showed no sign of contamination by aerolysin (lane a), a lower molecular weight form corresponding to aerolysin could be observed upon interaction with the granulocytes. A higher molecular weight band could also be observed upon incubation at 37 °C corresponding to the aerolysin heptamer (Fig. 2). These results agree well with our previous observations that proaerolysin can be converted into aerolysin by proteases provided by the host cell (14Abrami L. Fivaz M. Buckley J.T. Parton R.G. van der Goot F.G. J. Cell. Biol. 1998; 140: 525-540Crossref PubMed Scopus (195) Google Scholar). 2▪. Abrami, M. Fivaz, and F. G. van der Goot, unpublished data. These observations show that proaerolysin and aerolysin are able to bind to HL-60 cells and that the cells express proteases that can process the protoxin to its mature form. This allows heptamerization of the toxin and channel formation thereby leading to efflux of intracellular potassium, presumably to concomitant sodium entry, and membrane depolarization. To investigate whether the interaction of aerolysin with myeloid cells led to changes in cytosolic free Ca2+ concentration ([Ca2+]c), we exposed fura-2 loaded HL-60 promyelocytes and HL-60 granulocytes to either proaerolysin or trypsin-activated aerolysin (Fig. 3). Both, the protoxin and the mature toxin induced elevation of [Ca2+]c in a dose-dependent manner and this in both cell types. The kinetics of the [Ca2+]c increase were, however, markedly faster when the mature toxin was added rather than its precursor as previously observed for membrane depolarization (Fig. 3,A and C versus B and D). Also, the effects of both the pro and the mature toxin were more pronounced on the differentiated granulocytic HL-60 cells then on the immature promyelocytic cells. No changes in [Ca2+]c could be observed upon addition of the hemolytically inactive G202C/I445C mutant when added either in the pro or the mature form (Fig. 3 D). A striking feature of the aerolysin-induced [Ca2+]c elevations was their complex kinetics. Rather than presenting a monophasic increase, as might be expected for the insertion of a pore into the membrane, the kinetics were multiphasic: an initial, relatively rapid phase of [Ca2+]c change was followed by a more sustained phase. Thus, the toxin-induced Ca2+ response in myeloid cells was dose-dependent, accelerated by preactivation of the toxin, and depended on the state of differentiation of the cells. As the responses were most pronounced in HL-60 granulocytes stimulated with the mature toxin, these conditions were used to further analyze the mechanisms underlying the complex [Ca2+]cresponse to aerolysin. To investigate the source of the aerolysin-induced [Ca2+]c elevations, we exposed HL-60 granulocytes to aerolysin in a Ca2+-free medium. Under these conditions only Ca2+ release from intracellular stores can be detected, but not Ca2+ influx across the plasma membrane. As shown in Fig. 4 A, aerolysin (100 ng/ml) was able to induce [Ca2+]c elevations in a Ca2+-free medium, demonstrating that the toxin triggered Ca2+ release from intracellular stores. The observed [Ca2+]c elevations had complex kinetics. An initial phase peaked and decayed after approximately 40–60 s. A prolonged phase which increased toward a plateau could be observed 1–3 min after toxin addition. The binding competent, insertion-incompetent mutant G202C/I445C did not induce Ca2+-release (Fig. 4 A). However, when added in excess, the mutant was able to preclude Ca2+ release in response to wild type aerolysin (Fig. 4 B) confirming that the two variants of the toxin have the same acceptor sites on the cell and that they are limited in number. Many agonists induce Ca2+ release from intracellular stores through phospholipase C (PLC)-mediated Ins(1,4,5)P3generation and Ins(1,4,5)P3-induced Ca2+release from intracellular stores. To test whether PLC activation is involved in the aerolysin-induced Ca2+ release from intracellular stores, we have used the PLC inhibitor U73122. As shown in Fig. 4 C, this compound inhibited the initial phase of the aerolysin-induced Ca2+ release, however, neither the late phase of the Ca2+ release, nor the Ca2+ influx observed after Ca2+ readdition were affected. For many granulocyte agonists, Ca2+ release from intracellular stores is due to a G-protein-mediated activation of phospholipase C. In contrast to what is observed on many other cellular systems, agonist-PLC coupling in leukocytes is generally mediated by pertussis toxin-sensitive G-proteins (26Krause K.H. Schlegel W. Wollheim C.B. Andersson T. Waldvogel F.A. Lew P.D. J. Clin. Invest. 1985; 76: 1348-1354Crossref PubMed Scopus (105) Google Scholar). We therefore investigated the effect of pertussis toxin pretreatment on aerolysin-induced Ca2+release in the HL-60 granulocytes. As shown in Fig. 5, pertussis toxin pretreatment inhibited the initial, rapid phase of aerolysin-induced Ca2+ release (Fig. 5 D), but not the second, slower, phase (Fig. 5 E), nor the calcium entry across the plasma membrane. These results demonstrate an activation of pertussis toxin-sensitive G-proteins through aerolysin. The results obtained with pertussis toxin (Fig. 5) and the PLC inhibitor (Fig. 4), however, also demonstrate that there is a second phase of Ca2+ release which does not involve the G-protein/PLC/Ins(" @default.
• W1994792016 created "2016-06-24" @default.
• W1994792016 creator A5022397275 @default.
• W1994792016 creator A5051985484 @default.
• W1994792016 creator A5060495901 @default.
• W1994792016 creator A5073688967 @default.
• W1994792016 date "1998-07-01" @default.
• W1994792016 modified "2023-09-30" @default.
• W1994792016 title "Aerolysin Induces G-protein Activation and Ca2+Release from Intracellular Stores in Human Granulocytes" @default.
• W1994792016 cites W1209169208 @default.
• W1994792016 cites W1225403746 @default.
• W1994792016 cites W1495221297 @default.
• W1994792016 cites W1564090547 @default.
• W1994792016 cites W1665071864 @default.
• W1994792016 cites W1733593391 @default.
• W1994792016 cites W1774320314 @default.
• W1994792016 cites W1878693307 @default.
• W1994792016 cites W1902550810 @default.
• W1994792016 cites W1904524180 @default.
• W1994792016 cites W1949010416 @default.
• W1994792016 cites W1978079806 @default.
• W1994792016 cites W1979166620 @default.
• W1994792016 cites W2016776155 @default.
• W1994792016 cites W2023178515 @default.
• W1994792016 cites W2038923813 @default.
• W1994792016 cites W2044149109 @default.
• W1994792016 cites W2046261864 @default.
• W1994792016 cites W2048745890 @default.
• W1994792016 cites W2055071063 @default.
• W1994792016 cites W2070651788 @default.
• W1994792016 cites W2075778395 @default.
• W1994792016 cites W2080323034 @default.
• W1994792016 cites W2090866517 @default.
• W1994792016 cites W2100837269 @default.
• W1994792016 cites W2111832767 @default.
• W1994792016 cites W2122714737 @default.
• W1994792016 cites W2140261228 @default.
• W1994792016 cites W2146768590 @default.
• W1994792016 cites W2148551687 @default.
• W1994792016 cites W2153799259 @default.
• W1994792016 cites W2158249297 @default.
• W1994792016 cites W21831443 @default.
• W1994792016 cites W2254869068 @default.
• W1994792016 doi "https://doi.org/10.1074/jbc.273.29.18122" @default.
• W1994792016 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9660770" @default.
• W1994792016 hasPublicationYear "1998" @default.
• W1994792016 type Work @default.
• W1994792016 sameAs 1994792016 @default.
• W1994792016 citedByCount "73" @default.
• W1994792016 countsByYear W19947920162012 @default.
• W1994792016 countsByYear W19947920162013 @default.
• W1994792016 countsByYear W19947920162014 @default.
• W1994792016 countsByYear W19947920162015 @default.
• W1994792016 countsByYear W19947920162016 @default.
• W1994792016 countsByYear W19947920162017 @default.
• W1994792016 countsByYear W19947920162018 @default.
• W1994792016 countsByYear W19947920162019 @default.
• W1994792016 countsByYear W19947920162020 @default.
• W1994792016 countsByYear W19947920162021 @default.
• W1994792016 countsByYear W19947920162022 @default.
• W1994792016 countsByYear W19947920162023 @default.
• W1994792016 crossrefType "journal-article" @default.
• W1994792016 hasAuthorship W1994792016A5022397275 @default.
• W1994792016 hasAuthorship W1994792016A5051985484 @default.
• W1994792016 hasAuthorship W1994792016A5060495901 @default.
• W1994792016 hasAuthorship W1994792016A5073688967 @default.
• W1994792016 hasBestOaLocation W19947920161 @default.
• W1994792016 hasConcept C104317684 @default.
• W1994792016 hasConcept C12554922 @default.
• W1994792016 hasConcept C174319367 @default.
• W1994792016 hasConcept C185592680 @default.
• W1994792016 hasConcept C55493867 @default.
• W1994792016 hasConcept C60987743 @default.
• W1994792016 hasConcept C79879829 @default.
• W1994792016 hasConcept C86803240 @default.
• W1994792016 hasConcept C95444343 @default.
• W1994792016 hasConceptScore W1994792016C104317684 @default.
• W1994792016 hasConceptScore W1994792016C12554922 @default.
• W1994792016 hasConceptScore W1994792016C174319367 @default.
• W1994792016 hasConceptScore W1994792016C185592680 @default.
• W1994792016 hasConceptScore W1994792016C55493867 @default.
• W1994792016 hasConceptScore W1994792016C60987743 @default.
• W1994792016 hasConceptScore W1994792016C79879829 @default.
• W1994792016 hasConceptScore W1994792016C86803240 @default.
• W1994792016 hasConceptScore W1994792016C95444343 @default.
• W1994792016 hasIssue "29" @default.
• W1994792016 hasLocation W19947920161 @default.
• W1994792016 hasOpenAccess W1994792016 @default.
• W1994792016 hasPrimaryLocation W19947920161 @default.
• W1994792016 hasRelatedWork W1549554643 @default.
• W1994792016 hasRelatedWork W2026264997 @default.
• W1994792016 hasRelatedWork W2065261206 @default.
• W1994792016 hasRelatedWork W2091941153 @default.
• W1994792016 hasRelatedWork W2097812111 @default.
• W1994792016 hasRelatedWork W2132261509 @default.
• W1994792016 hasRelatedWork W2153616445 @default.
• W1994792016 hasRelatedWork W3125436686 @default.
• W1994792016 hasRelatedWork W4312518195 @default.

• next