Oxidative Stress Inhibits Calpain Activity in Situ
1998; Elsevier BV; Volume: 273; Issue: 21 Linguagem: Inglês
10.1074/jbc.273.21.13331
ISSN1083-351X
AutoresRodney P. Guttmann, Gail V.W. Johnson,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoIn this study, the effects of oxidative stress on calpain-mediated proteolysis and calpain I autolysis in situ were examined. Calpain activity was stimulated in SH-SY5Y human neuroblastoma cells with the calcium ionophore, ionomycin. Calpain-mediated proteolysis of the membrane-permeable fluorescent substrateN-succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine-7-amido-4-methylcoumarin, as well as the endogenous protein substrates microtubule-associated protein 2, tau and spectrin, was measured. Oxidative stress, induced by addition of either doxorubicin or 2-mercaptopyridineN-oxide, resulted in a significant decrease in the extent of ionophore-stimulated calpain activity of both the fluorescent compound and the endogenous substrates compared with control, normoxic conditions. Addition of glutathione ethyl ester, as well as other antioxidants, resulted in the retention/recovery of calpain activity, indicating that oxidation-induced calpain inactivation was preventable/reversible. The rate of autolytic conversion of the large subunit of calpain I from 80 to 78 to 76 kDa was decreased during oxidative stress; however, the extent of calpain autolysis was not altered. These data indicate that oxidative stress may reversibly inactivate calpain I in vivo. In this study, the effects of oxidative stress on calpain-mediated proteolysis and calpain I autolysis in situ were examined. Calpain activity was stimulated in SH-SY5Y human neuroblastoma cells with the calcium ionophore, ionomycin. Calpain-mediated proteolysis of the membrane-permeable fluorescent substrateN-succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine-7-amido-4-methylcoumarin, as well as the endogenous protein substrates microtubule-associated protein 2, tau and spectrin, was measured. Oxidative stress, induced by addition of either doxorubicin or 2-mercaptopyridineN-oxide, resulted in a significant decrease in the extent of ionophore-stimulated calpain activity of both the fluorescent compound and the endogenous substrates compared with control, normoxic conditions. Addition of glutathione ethyl ester, as well as other antioxidants, resulted in the retention/recovery of calpain activity, indicating that oxidation-induced calpain inactivation was preventable/reversible. The rate of autolytic conversion of the large subunit of calpain I from 80 to 78 to 76 kDa was decreased during oxidative stress; however, the extent of calpain autolysis was not altered. These data indicate that oxidative stress may reversibly inactivate calpain I in vivo. Calpains are a family of calcium-dependent thiol proteases that require both calcium (1Murachi T. Biochem. Int. 1989; 18: 263-294PubMed Google Scholar) and a reduced environment (2Guttmann R.P. Elce J.S. Bell P.D. Isbell J.C. Johnson G.V.W. J. Biol. Chem. 1997; 272: 2005-2012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) for activity. These proteolytic enzymes have been postulated to play a role in many physiological processes (3Lynch G. Baudry M. Brain Res. Bull. 1987; 18: 809-815Crossref PubMed Scopus (111) Google Scholar, 4Pontremoli S. Melloni E. Annu. Rev. Biochem. 1986; 55: 455-481Crossref PubMed Scopus (204) Google Scholar, 5Schollmeyer J.E. Science. 1988; 240: 911-913Crossref PubMed Scopus (131) Google Scholar, 6Melloni E. Pontremoli S. Trends Neurosci. 1989; 12: 438-444Abstract Full Text PDF PubMed Scopus (175) Google Scholar) and disease states (7Neumar R.W. Hagle S.M. DeGracia D.J. Krause G.S. White B.C. J. Neurochem. 1996; 66: 421-424Crossref PubMed Scopus (68) Google Scholar, 8Blomgren K. Kawashima S. Saido T.C. Karlsson J.O. Elmered A. Hagberg H. Brain Res. 1995; 684: 143-149Crossref PubMed Scopus (50) Google Scholar, 9Saito K.I. Elce J.S. Hamos J.E. Nixon R.A. Proc. 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Although homologous, calpains I and II require different concentrations of calcium for activity in vitro. Calpain II requires 200–1000 μm calcium (15Cong J. Goll D.E. Peterson A.M. Kapprell H.-P. J. Biol. Chem. 1989; 264: 10096-10103Abstract Full Text PDF PubMed Google Scholar), and calpain I requires 3–50 μm calcium (15Cong J. Goll D.E. Peterson A.M. Kapprell H.-P. J. Biol. Chem. 1989; 264: 10096-10103Abstract Full Text PDF PubMed Google Scholar) for half-maximal activity. Although such high calcium concentrations have been demonstrated in the presynaptic terminals of neurons (16Llinas R. Sugimori M. Silver R.B. Science. 1992; 256: 677-679Crossref PubMed Scopus (707) Google Scholar) and under pathological conditions (17Kudo Y. Ogura A. Br. J. Pharmacol. 1986; 89: 191-198Crossref PubMed Scopus (202) Google Scholar), it seems clear that physiological calcium concentrations (100–800 nm (18Jonas E.A. Knox R.J. Smith T.C. Wayne N.L. Connor J.A. Kaczmarek L.K. Nature. 1997; 385: 343-346Crossref PubMed Scopus (109) Google Scholar)) are sufficient to activate calpain I (19Song D.K. Malmstrom T. Kater S.B. Mykles D.L. J. Neurosci. Res. 1994; 39: 474-481Crossref PubMed Scopus (45) Google Scholar, 20Zhang W. Lane R.D. Mellgren R.L. J. Biol. Chem. 1996; 271: 18825-18830Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 21Molinari M. Anagli J. Carafoli E. J. Biol. Chem. 1994; 269: 27992-27995Abstract Full Text PDF PubMed Google Scholar, 22Salamino F. De Tullio R. Menhotti P. Viotti P.L. Melloni E. Pontremoli S. Biochem. J. 1993; 290: 191-197Crossref PubMed Scopus (41) Google Scholar). The focus of this study was calpain I because it is present in neurons and has been postulated to play a role in neuronal death associated with ischemia (7Neumar R.W. Hagle S.M. DeGracia D.J. Krause G.S. White B.C. J. Neurochem. 1996; 66: 421-424Crossref PubMed Scopus (68) Google Scholar, 8Blomgren K. Kawashima S. Saido T.C. Karlsson J.O. Elmered A. Hagberg H. Brain Res. 1995; 684: 143-149Crossref PubMed Scopus (50) Google Scholar) and certain neurodegenerative disorders (9Saito K.I. Elce J.S. Hamos J.E. Nixon R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2628-2632Crossref PubMed Scopus (534) Google Scholar, 10Bartus R.T. Elliott P.J. Hayward N.J. Dean R.L. Harbeson S. Straub J.A. Li Z. Powers J.C. Neurol. Res. 1995; 17: 249-258Crossref PubMed Scopus (109) Google Scholar, 11Nilsson E. Alafuzoff I. Blennow K. Blomgren K. Hall C.M. Janson I. Karlsson I. Wallin A. Gottfries C.G. Karlsson J.O. Neurobiol. Aging. 1990; 11: 425-431Crossref PubMed Scopus (61) Google Scholar, 12Nixon R.A. Saito K.I. Grynspan F. Griffin W.R. Katayama S. Honda T. Mohan P.S. Shea T.B. Beermann M. Ann. N. Y. Acad. Sci. 1994; 747: 77-91Crossref PubMed Scopus (240) Google Scholar). Calpain I is a heterodimer composed of a unique 80-kDa catalytic subunit and a 30-kDa, regulatory subunit common to both calpain I and II. Calpain undergoes calcium-dependent autolysis, a multi-step, self-proteolytic event that removes a number of amino acids from the N terminus of each subunit resulting in increased proteolytic activity and increased sensitivity to calcium (23Goll D.E. Thompson V.F. Taylor R.G. Zalewska T. BioEssays. 1992; 14: 549-556Crossref PubMed Scopus (186) Google Scholar). Autolysis results in the conversion of the 80-kDa subunit to a 76-kDa form through a 78-kDa intermediate (24Zimmerman U.-J.P. Schlaeper W.W. Biochemistry. 1982; 21: 3977-3983Crossref PubMed Scopus (138) Google Scholar) which occurs prior to the conversion of the 30-kDa subunit regulatory to 18 kDa (25Dayton W.R. Biochim. Biophys. Acta. 1982; 709: 166-172Crossref PubMed Scopus (65) Google Scholar). Although still controversial, there is substantial evidence to indicate that non-autolyzed form of calpain I is active under physiological conditions (10Bartus R.T. Elliott P.J. Hayward N.J. Dean R.L. Harbeson S. Straub J.A. Li Z. Powers J.C. Neurol. Res. 1995; 17: 249-258Crossref PubMed Scopus (109) Google Scholar, 21Molinari M. Anagli J. Carafoli E. J. Biol. Chem. 1994; 269: 27992-27995Abstract Full Text PDF PubMed Google Scholar, 26Suzuki K. Sorimachi H. Yoshizawa T. Kinbara K. Ishiura S. Biol. Chem. Hoppe-Seyler. 1995; 376: 523-529Crossref PubMed Scopus (211) Google Scholar). The disparate findings concerning the proteolytic activity of the native 80-kDa form of calpain I may be due to the process of autolysis being modulated by more than calcium concentration alone (e.g. the presence of phospholipids, calpastatin, or other unknown factors) (23Goll D.E. Thompson V.F. Taylor R.G. Zalewska T. BioEssays. 1992; 14: 549-556Crossref PubMed Scopus (186) Google Scholar). Additionally, it is also unclear as to the specific role of the 30-kDa non-catalytic subunit in modulating calpain autolysis (27Cottin P. Poussard S. Desmazes J.P. Georgescauld D. Ducastaing A. Biochim. Biophys. Acta. 1991; 1079: 139-145Crossref PubMed Scopus (32) Google Scholar, 28Saido T.C. Nagao S. Shiramine M. Tsukaguchi M. Yoshizawa T. Sorimachi H. Ito H. Tsuchiya T. Kawashima S. Suzuki K. FEBS Lett. 1994; 346: 263-267Crossref PubMed Scopus (63) Google Scholar). Calpain activity can be modulated by redox state, characterized by decreased activity, in vitro, in the presence of oxidant (2Guttmann R.P. Elce J.S. Bell P.D. Isbell J.C. Johnson G.V.W. J. Biol. Chem. 1997; 272: 2005-2012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The reason for this is likely due to the mechanism by which calpain I hydrolyzes a peptide bond. For proteolysis to proceed, a transfer of electrons between the specific cysteine, histidine, and arginine residues within the catalytic triad of the active site (29Mehdi S. Trends Biochem. Sci. 1991; 16: 150-153Abstract Full Text PDF PubMed Scopus (196) Google Scholar) must occur. This requires that these amino acid residues be maintained in a properly charged state that can be modulated by their local microenvironment. Of the amino acids in the catalytic triad, it is the cysteine residue that is the most susceptible to oxidative inactivation (30Neumann N.P. Methods Enzymol. 1972; 25: 393-400Crossref PubMed Scopus (75) Google Scholar) and is therefore likely to play a significant role in decreased calpain I activity upon exposure to an oxidizing environment. The effects of an oxidizing environment on calpain I activity are important to understand because oxidative stress has been linked to pathological states in which calpain I has been suggested to play a role, including Alzheimer's disease (31Choi B.H. Neurobiol. Aging. 1995; 16: 675-678Crossref PubMed Scopus (43) Google Scholar, 32Benzi G. Moretti A. Neurobiol. Aging. 1995; 16: 661-674Crossref PubMed Scopus (351) Google Scholar) and ischemia (33Bartus R.T. Baker K.L. Heiser A.D. Sawyer S.D. Dean R.L. Elliott P.J. Straub J.A. J. Cereb. Blood Flow Metab. 1994; 14: 537-544Crossref PubMed Scopus (165) Google Scholar, 34Halliwell B. J. Neurochem. 1992; 59: 1609-1623Crossref PubMed Scopus (2666) Google Scholar). In the present study, both calpain I autolysis and proteolytic activity were examined in situ, under control and oxidatively stressed conditions. These results indicate that calpain I activity stimulated by increasing intracellular calcium can be inhibited by oxidizing conditions. Additionally, oxidative inactivation was prevented by incubation with membrane-permeable antioxidants. Finally, oxidative stress decreased the rate of autolytic conversion of calpain I from 80 to 78 to 76 kDa, but the extent was unaffected. Ionomycin, glutathione-ethyl ester (GSH), 1The abbreviations used are: GSH, glutathione ethyl ester; CBZ, benzyloxycarbonyl; Z-LLY-CHN2,N-CBZ-l-leucyl-l-leucyl-l-tyrosine, diazomethyl ketone; FF1, FotoFenton1 or 2-mercaptopyridineN-oxide; DCFH2-DA, 2′,7′-dichlorodihydrofluorescein diacetate; Suc-LLVY-AMC,N-succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine-7-amido-4-methylcoumarin; DCF, dichlorofluorescein; MAP-2, microtubule-associated protein 2. propyl gallate, poly-l-lysine, and retinoic acid were from Sigma;N-CBZ-l-leucyl-l-leucyl-l-tyrosine-diazomethyl ketone (Z-LLY-CHN2), 2-mercaptopyridine N-oxide, also known as FotoFenton1 (FF1), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA) were from Molecular Probes. Fura-2 was from Teflabs, and the fluorescent peptideN-succinyl-l-leucyl-l-leucyl-l-valyl-l-tyrosine-7-amido-4-methylcoumarin (Suc-LLVY-AMC) was from Bachem. Trolox was from Merck. Phenylmethanesulfonyl fluoride and pepstatin were from Boehringer Mannheim. Ac-Tyr-Val-Ala-Asp-CMK (caspase inhibitor 2), CBZ-Phe-Gly-NHO-Bz, and CBZ-Phe-Gly-NHO-Bz-pOMe were from Calbiochem. All other reagents were of the highest grade possible. All calpain assays were carried out at 37 °C in a humidified 5% CO2 incubator. SH-SY5Y human neuroblastoma cells were plated on 60-mm Corning/Costar dishes and differentiated with 20 μm retinoic acid for 6–8 days in RPMI 1640 media supplemented with 10% horse serum, 5% fetal clone II, 1 mm glutamine, and 100 units/ml penicillin/streptomycin. Cells were at approximately 70–80% confluence in a monolayer at the time of experimentation. Free calcium concentrations were determined using cells attached to poly-l-lysine-coated glass coverslips (Corning) with the calcium indicator dye, fura-2. Prior to experimentation, cells were loaded with 5 μmacetoxymethyl ester form of fura-2. Coverslips were placed in an imaging chamber (Warner Instrument Co.) and mounted in a heater platform on the stage of a Nikon Diaphot. The cells were maintained at 37 °C in a Ringer solution for the duration of the experiments. Fura-2 was excited at alternating wavelengths of 340- and 380-nm using a 75-watt xenon light source and a filter wheel (Ionoptix system, Ionoptix Corp., Milton, MA). Emitted wavelengths passed through a 510-nm filter cube set before detection by an enhanced CCD camera. Data were stored and processed using IonWizard software. Calibration of fura-2 fluorescence was performed per standard protocols where the ratio (R) of emitted signals at 340- and 380-nm excitation wavelengths provided an index of calcium concentration, which was estimated according to the equation: [Ca2+]free = K d ·S f/S b· (R/R min)/(R max/R), where K d is the effective dissociation constant of fura-2, R min and R max are the 340- to 380-nm ratios, and S f andS b are fluorescence values in the absence and presence of saturating calcium, respectively (35Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). TheK d value of fura-2 for calcium was estimated to be 240 nm (35Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Calpain activity was measured by immunoblot analysis of endogenous substrates or proteolytic cleavage of the fluorescent calpain substrate Suc-LLVY-AMC under control and oxidizing conditions. To oxidatively stress the cells, doxorubicin was added to a final concentration of 5 μm for 24 h before ionomycin treatment (36Sarvzyan N. Am. J. Physiol. 1996; 271 (79–H2O85): H2OCrossref Google Scholar), while FF1 was used at 1 mm immediately prior to ionomycin addition. Doxorubicin, an anthracycline antibiotic, has been shown to generate superoxide radicals through redox autoxidation of the quinone group that acts on the mitochondrion (37Goodman J. Hochstein P. Biochem. Biophys. Res. Commun. 1977; 77: 797-803Crossref PubMed Scopus (526) Google Scholar). Production of free radicals by FF1 occurs, in the presence of UV light, resulting from photodecomposition of the compound that generates hydroxyl radicals (38Epe B. Ballmaier D. Adam W. Grimm G.N. Saha-Moller C.R. Nucleic Acids Res. 1996; 24: 1625-1631Crossref PubMed Scopus (51) Google Scholar). The antioxidants GSH (5 mm), Trolox (100 μm), or propyl gallate (5 μm) were added at the same time as oxidant (either 24 h or immediately prior to ionomycin treatment). After 24 h, cells were rinsed once in serum-free RPMI 1640 media containing the appropriate treatment to maintain experimental conditions, and then 3 ml of serum-free media were added, also containing the treatment compounds. Cells were treated with 2 μm ionomycin for the times indicated, rinsed once with phosphate-buffered saline, and collected in 2× Laemmli stop buffer without dye. Samples were immediately heated to 100 °C for 10 min, sonicated, and protein concentrations determined by the Lowry method, after acid precipitation of the protein. Bromphenol blue was added to the samples, and 10 or 20 μg of total protein were separated on SDS-polyacrylamide gels (4–12.5% for MAP-2 and 6.5% for calpain I, 10% for tau, 8% for calpastatin, and 5% spectrin), transferred to nitrocellulose (39Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar), and immunoblotted with a monoclonal antibody to either MAP-2 (AP14, a gift from Dr. L. Binder), calpain I (a gift from Dr. J. Elce), tau (5A6 (40Johnson G.V. Seubert P. Cox T.M. Motter R. Brown J.P. Galasko D. J. Neurochem. 1997; 68: 430-433Crossref PubMed Scopus (140) Google Scholar); Tau-5, a gift from Dr. L. Binder (41Carmel G. Mager E.M. Binder L.I. Kuret J. J. Biol. Chem. 1996; 271: 32789-32795Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar), combination), calpastatin (a gift from Dr. J. Glass), or a polyclonal antibody to spectrin (Ab992, Chemicon). After incubation with the primary antibody, the blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody and developed with 3,3-diaminobenzidine in the presence of hydrogen peroxide or by enhanced chemiluminescence (Amersham Pharmacia Biotech) per manufacturer's instructions. The resulting immunoblots were quantitated using a Bio-Rad imaging densitometer (model GS-670). Data were evaluated using analysis of variance, and values were considered significantly different when p < 0.05. In the case of experiments utilizing the fluorescence compound, SH-SY5Y cells were similarly prepared and treated on 24-well Corning/Costar plates. Prior to addition of ionomycin, cells were loaded with 80 μm Suc-LLVY-AMC (42Bronk S.F. Gores G.J. Am. J. Physiol. 1993; 264: G744-G751PubMed Google Scholar) for 20 min at 37 °C in a humidified 5% CO2 incubator. Proteolysis of the fluorescent probe was monitored using a fluorescent plate reading system (Bio-Tek FL500) with filter settings of 360 ± 20 nm for excitation and 470 ± 20 nm for emission. Fluorescent readings were made every 20 min for up to 1 h. Cells were replaced in the incubator between readings to maintain both temperature and pH for optimal cell viability. The hydrogen peroxide-sensitive fluorescent compound DCFH2-DA was used to indicate levels of oxidative stress induced by either doxorubicin or FF1. DCFH2-DA is a well established indicator of intracellular hydrogen peroxide formation which, after intracellular deacylation, remains non-fluorescent but is transformed into the fluorescent form (dichlorofluorescein, DCF) via oxidation (43Rosenkranz A.R. Schmaldienst S. Stuhlmeier K.M. Chen W. Knapp W. Zlabinger G.J. J. Immunol. Methods. 1992; 156: 39-45Crossref PubMed Scopus (480) Google Scholar). Cells were incubated with DCFH2-DA 10 min after the addition of ionomycin for all treatment conditions. Fluorescent readings were then taken every 20 min for up to 60 min. Fluorescence was detected with the FL500 (Bio-Tek) fluorescence plate reader with filter wheels set at 485 ± 20 nm and 530 ± 25 nm for excitation and emission, respectively. The release of the intracellular enzyme, lactate dehydrogenase, into the media was used as a quantitative measure of cell viability. Adherent SH-SY5Y cells on 24-well Corning/Costar dishes were differentiated, rinsed, treated, and stimulated with ionomycin as described above. The measurement of lactate dehydrogenase release was performed as described previously (44Davis P.K. Dudek S.M. Johnson G.V.W. J. Neurochem. 1997; 68: 2338-2347Crossref PubMed Scopus (38) Google Scholar). To estimate the relative amount of free radical production in response to doxorubicin or FF1, the formation of the fluorescent oxidized product, DCF from DCFH2-DA, was measured (Fig. 1). Treatment of SH-SY5Y cells with FF1 resulted in a significant increase in peroxide production as indicated by an increase in DCF production. Although doxorubicin has been clearly demonstrated to be a potent inducer of oxidative stress (45MacIntosh L.J. Sapolosky R.M. Exp. Neurol. 1996; 141: 201-206Crossref PubMed Scopus (189) Google Scholar), treatment with doxorubicin did not significantly increase DCF production. This is probably due to the fact that DCFH2-DA is a selective indicator of hydrogen peroxide formation (46Royall J.A. Ischiropoulos H. J. Biochem. Biophys. 1993; 302: 348-355Crossref Scopus (1050) Google Scholar, 47Zhu H. Bannenberg G.L. Moldeus P. Shertzer H.G. Arch. Toxicol. 1994; 68: 582-587Crossref PubMed Scopus (251) Google Scholar) and does not react well with superoxide anions which are the predominant free radical generated in response to doxorubicin treatment (37Goodman J. Hochstein P. Biochem. Biophys. Res. Commun. 1977; 77: 797-803Crossref PubMed Scopus (526) Google Scholar, 46Royall J.A. Ischiropoulos H. J. Biochem. Biophys. 1993; 302: 348-355Crossref Scopus (1050) Google Scholar). When the antioxidants propyl gallate, Trolox, or GSH were included with either FF1 or doxorubicin, a significant decrease in DCF production was observed. Antioxidants alone also significantly reduced the hydrogen peroxide concentration to below the level observed in control cells. The fluorescent calpain-selective substrate peptide Suc-LLVY-AMC (42Bronk S.F. Gores G.J. Am. J. Physiol. 1993; 264: G744-G751PubMed Google Scholar) was used to measure calpain activity in SH-SY5Y cells. As in studies by other investigators (48Kavita U. Mizel S.B. J. Biol. Chem. 1995; 270: 27758-27765Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 49Norris F.A. Atkins R.C. Majerus P.W. J. Biol. Chem. 1997; 272: 10987-10989Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 50Schoenwaelder S.M. Yuan Y. Cooray P. Salem H.H. Jackson S.P. J. Biol. Chem. 1997; 272: 1694-16702Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) calpain activity was stimulated through the use of a calcium ionophore. Cells were treated with 2 μm ionomycin, and the increase in fluorescence measured at 20-min intervals for up to 60 min. Ionomycin increased the intracellular calcium concentration from the 50–80 nm basal concentration to 700–800 nm. Maximal calcium concentration was reached after 8–9 min of exposure and remained elevated for the duration of the experiments (data not shown). Cells remained adherent over the time course, with no loss of viability as indicated by lack of increase in lactate dehydrogenase release (data not shown). Fig. 2 shows the increase in Suc-LLVY-AMC degradation, indicated by increased fluorescence, in response to the various treatments relative to conditions in the presence of ionophore alone (control) after 40 min. Proteolysis of the fluorescent substrate was significantly inhibited by the presence of either doxorubicin or FF1 to approximately 35% of ionomycin treatment alone. When cells were treated with FF1 in the presence of GSH, all oxidant-induced inhibition of calpain activity was prevented. GSH also significantly ameliorated the inhibition of calpain activity in response to doxorubicin. In addition, Trolox or propyl gallate significantly reduced the extent of calpain inactivation by the oxidants but to lesser extent than GSH. GSH treatment significantly increased ionomycin-stimulated calpain proteolysis of the fluorescent probe compared with ionomycin treatment under normoxic conditions. Because the fluorescent probe Suc-LLVY-AMC is selective but not totally specific for calpain-mediated proteolysis (2Guttmann R.P. Elce J.S. Bell P.D. Isbell J.C. Johnson G.V.W. J. Biol. Chem. 1997; 272: 2005-2012Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 42Bronk S.F. Gores G.J. Am. J. Physiol. 1993; 264: G744-G751PubMed Google Scholar, 51Sasaki T. Kikuchi T. Yumoto N. Yoshimura N. Murachi T. J. Biol. Chem. 1984; 259: 12489-12494Abstract Full Text PDF PubMed Google Scholar), experiments were carried out using the calpain-selective protease inhibitors Z-LLY-CHN2 (42Bronk S.F. Gores G.J. Am. J. Physiol. 1993; 264: G744-G751PubMed Google Scholar, 52Mellgren R.L. Shaw E. Mericle M.T. Exp. Cell Res. 1994; 215: 164-171Crossref PubMed Scopus (34) Google Scholar, 53Mellgren R.L. J. Biol. Chem. 1997; 272: 29899-29903Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) or calpain inhibitor I (54Saito K. Nixon R.A. Neurochem. Res. 1993; 18: 231-233Crossref PubMed Scopus (22) Google Scholar) to determine the amount of Suc-LLVY-AMC proteolysis which is not calpain-dependent. Addition of either 25 μmZ-LLY-CHN2 (Fig. 2) or 20 μm calpain inhibitor I (data not shown) inhibited ionomycin-stimulated proteolysis of the fluorescent compound by greater than 90%, suggesting that the majority of the increase in fluorescence in response to ionomycin is due to calpain. Because these two inhibitors may also inhibit cathepsin L (55Crawford C. Mason R.W. Wikstrom P. Shaw E. Biochem. J. 1988; 253: 751-758Crossref PubMed Scopus (126) Google Scholar, 56Anagli J. Hagmann J. Shaw E. Biochem. J. 1991; 274: 497-502Crossref PubMed Scopus (27) Google Scholar), additional experiments were carried out to eliminate the possibility that cathepsin L was contributing to proteolysis of Suc-LLVY-AMC. Inhibition of cathepsin L activity with either of the cathepsin L-specific inhibitors CBZ-Phe-Gly-NHO-Bz (20 μm) or CBZ-Phe-Gly-NHO-Bz-pOMe (20 μm) (57Demuth H.U. Schierhorn A. Bryan P. Hofke R. Kirschke H. Bromme D. Biochim. Biophys. Acta. 1996; 1295: 179-186Crossref PubMed Scopus (19) Google Scholar) resulted in no decrease in ionophore-stimulated proteolysis of the fluorescent compound. In addition, treatment of SH-SY5Y cells with other non-calpain inhibitors (phenylmethanesulfonyl fluoride (0.1 mm), pepstatin (1 μg/ml), or Ac-Tyr-Val-Ala-Asp-CMK (caspase inhibitor 2, 1 μg/ml)) also did not result in inhibition of ionomycin-stimulated proteolysis of Suc-LLVY-AMC (data not shown), further suggesting that the observed proteolytic activity is due to calpain activation. The effects of oxidative stress on the proteolysis MAP-2 by calpain were examined. MAP-2 has been identified previously both in vitro (58Johnson G.V.W. Litersky J.M. Jope R.S. J. Neurochem. 1991; 56: 1630-1638Crossref PubMed Scopus (171) Google Scholar) and in situ (8Blomgren K. Kawashima S. Saido T.C. Karlsson J.O. Elmered A. Hagberg H. Brain Res. 1995; 684: 143-149Crossref PubMed Scopus (50) Google Scholar), as a highly sensitive calpain substrate. SH-SY5Y cells were incubated with 2 μm ionomycin for 10 min, and the extent of MAP-2 degradation was quantitated by immunoblot analysis. Fig.3 shows a representative immunoblot and quantitative analysis of ionophore-stimulated MAP-2 degradation. The presence of either doxorubicin or FF1 resulted in significant reduction in MAP-2 degradation, similar to the inhibition observed with the fluorescent compound (Fig. 2). Also, in good agreement with the fluorescent data was the finding that oxidation-induced inhibition of MAP-2 degradation was prevented by addition of the antioxidant GSH. GSH treatment alone of ionomycin-stimulated cells, however, had no significant effect on MAP-2 proteolysis compared with ionomycin treatment under normoxic conditions (Fig. 3). The second endogenous substrate that was studied was the microtubule-associated protein tau. Tau has been demonstrated to be an excellent substrate of calpain both in vitro (59Johnson G.V.W. Jope R.S. Binder L.I. Biochem. Biophys. Res. Commun. 1989; 163: 1505-1511Crossref PubMed Scopus (148) Google Scholar, 60Mercken M. Grynspan F. Nixon R.A. FEBS Lett. 1995; 368: 10-14Crossref PubMed Scopus (74) Google Scholar) andin situ (61Kampfl A. Zhao X. Whitson J.S. Posmantur R. Dixon C.E. Yang K. Clifton G.L. Hayes R.L. Eur. J. Neurosci. 1996; 8: 344-352Crossref PubMed Scopus (25) Google Scholar). Fig. 4 shows a representative immunoblot and quantitative analysis of tau degradation after the cells were treated for 15 min with ionomycin. Consistent with the previous findings, calpain degradation of tau was significantly inhibited by oxidative stress with both FF1 and doxorubicin. The inhibition of calpain, by either oxidative stress inducer, was similar to that observed with 25 μm Z-LLY-CHN2. The last endogenous substrate studied was spectrin, another well established calpain substrate in vitro (58Johnson G.V.W. Litersky J.M. Jope R.S. J. Neurochem. 1991; 56: 1630-1638Crossref PubMed Scopus (171) Google Scholar) and in vivo (62Bednarski E. Vanderklish P. Gall C. Saido T.C. Bahr B.A. Lynch G. Brain Res. 1995; 694: 147-157Crossref PubMed Scopus (61) Google Scholar), although less sensitive than MAP-2 (8Blomgren K. Kawashima S. Saido T.C. Karlsson J.O. Elmered A. Hagberg H. Brain Res. 1995; 684: 143-149Crossref PubMed Scopus (
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