Inversion of the Intracellular Na+/K+Ratio Blocks Apoptosis in Vascular Smooth Muscle at a Site Upstream of Caspase-3

• DOI: 10.1074/jbc.274.23.16545
• Publication Date: 2002-07-26T15:11:54Z
• Authors: Sergei N. Orlov; Nathalie Thorin-T...; Sergei V. Kotelev...; Johanne Tremblay; Pavel Hamet
• Keywords: Male; 0303 health sciences; L-Lactate Dehydrogenase; Caspase 3; Sodium; Apoptosis; Muscle, Smooth, Vascular; Rats; Jurkat Cells; 03 medical and health sciences; Caspases; Potassium; Animals; Humans; Enzyme Inhibitors; Ouabain; Cell Size

Long term elevation of the intracellular Na+/K+ ratio inhibits macromolecule synthesis and proliferation in majority cell types studied so far, including vascular smooth muscle cells (VSMC). We report here that inhibition Na+,K+ pump VSMC by ouabain or a 1-h preincubation K+-depleted medium attenuated apoptosis triggered serum withdrawal, staurosporine, okadaic acid. In absence ouabain, both DNA degradation Caspase-3 activation undergoing were insensitive to modification extracellular as well hyperosmotic shrinkage. contrast, protection from was abolished under equimolar substitution Na+o with K+o, showing antiapoptotic action caused inversion ratio. Unlike VSMC, same level increment [Na+]i/[K+]i 2-h Jurkat did not affect chromatin cleavage activity treatment Fas ligand, Thus, our results show for first time similar proliferation, maintenance physiologically low Na+/K+ratio is required progression apoptosis. The transmembrane gradient monovalent cation (high [K+]i [Na+]i) universal property all nucleated cells, its dissipation viewed hallmark necrotic-type death (1Wyllie A.H. Bowen I.D. Lockshin R.A. Cell Death Biology Pathology. Chapman Hall, London1981: 9-34Crossref Google Scholar, 2Cotter T.G. Lennon S.V. Glynn J.G. Martin S.J. Cancer Res. 1990; 10: 1153-1160Google Scholar). It shown transient moderate rise Na+concentration mitogen-treated involved rejoining strand breaks preceding (3Prasad C.V. Greer W.L. Severini A. Kaplan 1987; 47: 5397-5400PubMed Scholar), whereas long blocks macromolecular cycle eukaryotic far (4Lubin M. Nature. 1967; 213: 451-453Crossref PubMed Scopus (167) 5Kaplan Annu. Rev. Physiol. 1978; 40: 19-41Crossref (170) 6Lubin Biochem. Biophys. Commun. 1980; 97: 1060-1067Crossref (29) 7Brodie C. Tordai Saloga J. Domenico Gelfand E.W. Cell. 1995; 165: 246-253Crossref (28) (VSMC) 1The abbreviations used are: cell(s); DMEM, Dulbecco's modified Eagle's medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CHO, Chinese hamster ovary; AMC, 7-amino-4-methylcoumarin; YVAD, N-acetyl-Tyr-Val-Ala-Asp; DEVD, N-acetyl-Asp-Glu-Val-Asp. (8Henningsen N. Stavenow L. Borg Scand. Clin. Lab. Invest. 1984; 44: 197-201Crossref (4) 9Bobik Grooms Grinpukel S. Little P.J. Hypertens. 1988; 6 Suppl. 4: S219-S221Crossref Much less known about role ions triggering programmed (apoptosis). shrinkage one initial morphological markers particularly (10Orlov S.N. Dam T.V. Tremblay Hamet P. 1996; 221: 708-715Crossref (60) immune system apoptotic impressive "shrinkage-mediated necrosis" originally proposed describe this type (11Kerr J.F.R. Wyllie Currie A.R. Br. Cancer. 1972; 26: 239-257Crossref (12858) striking increase density shrunken separate intact (12Ohyama H. Yamada T. Watanabe I. Radiat. 1981; 85: 333-339Crossref (41) Scholar,13Klassen N.V. Walker P.R. Ross C.K. Cygler Lach B. Int. Biol. 1993; 64: 571-581Crossref (53) lymphocytes, volume decrease loss potassium chloride (14Bortner C.D. Hughes Jr., F.M. Cidlowski J.A. Chem. 1997; 272: 32436-32442Abstract Full Text PDF (509) Scholar) major organic osmolyte, taurine (15Lang F. Madlung Uhlemann A.C. Risler Gulbins E. Pfluegers Arch. Eur. 1998; 436: 377-383Crossref (71) because CD95 receptor-mediated Cl− K+ channels outward transporter (for recent review, see Ref. 16Lang Lepple-Wienhues Szabo Siemen D. Lang Volume Regulation. Contributions Nephrology. Karger, Basel1998: 158-168Google However, involvement perturbation ion composition ionic strength cytoplasm development machinery remains unclear. Recently, it Na+ protects induced ligand receptors suggesting K+gradients plays cells. Here, we contrast at site upstream Caspase-3, independently cations volume. obtained explant methods aortas 10–13-week-old male rats described previously (17Franks D.J. Plamondon 119: 41-45Crossref (67) cultured DMEM 10% calf serum, 100 units/ml penicillin, μg/ml streptomycin, between 10 16 passages. Cells transfected c-myc (VSMC-MYC) E1A adenovirus (VSMC-E1A) accordance protocol (18Bennett M.R. Evan G.I. Newby Circ. 1994; 74: 525-536Crossref (185) 19Bennett Schwartz S.M. 77: 266-273Crossref (145) addition 500 Geneticin. American Type Culture Collection RPMI 1640 supplied antibiotics, sodium pyruvate, glutamate, β-mercaptoethanol. Chromatin estimated technique detail Briefly, grown 24-well dishes labeled 24 h serum-supplied [3H]thymidine (1 μCi/ml), washed 2 × ml incubated DMEM. For 48 h, pretreated indicated Fig. Table III, twice serum-containing medium, 0.5 without containing different inducers To measure content fragments, transferred on ice, 1 ice-cold lysis buffer (10 mm EDTA, Tris-HCl, 0.5% Triton X-100 (pH 8.0)) added. 15 min, lysate Eppendorf tubes sedimented (12,000 rpm, min), supernatant measurement radioactivity (A 1t). remaining pellets wells extracted mixture 1% SDS 4 combined, counted 2). quantified fragments normalized total of3H-labeled equation (1.5 1t −A 1o)/(A +A − 1.5A 1o)) 100%, whereA 1o value ofA before induction fragmentation [3H]thymidine-labeled resuspended 106 cells/ml serum; additions are IV. At intervals 4, μl suspension mixed phosphate-buffered saline X-100, measured (20Garner R. Helgason Atkinson E.A. Pinkoski M.J. Ostergaard H.L. Sorensen O. Fu Lapchak P.H. Rabinovitch McElhaney J.E. Berke G. Bleackley R.C. Immunol. 153: 5413-5420PubMed Scholar).Table IIIEffect Na + K control ouabain-treated VSMC-E1AIntracellular cationsChromatin fragmentsConcentration Na+and incubation mediumControl cellsOuabain-treated cellsControl cellsNa+iK+iNa+iK+i10% CSCS free10% freemmnmol/mg protein%%1. [Na+]o = 137; [K+]o= 542 ± 14297 36312 47ap < 0.001 compared ouabain-untreated cells.99 21ap cells.5.2 0.632.8 4.81.9 0.5ap cells.6.3 1.4ap cells.2. 14; [K+]o 12838 11311 4051 18293 365.9 1.034.6 5.05.0 0.832.0 5.5To estimate cleavage, presence (CS) solution (DMEM, [Na+] 137 mm; [K+] 5 line 1) NaCl KCl ([Na+] 14 128 2) then an additional media CS. Intracellular steady-state distribution isotopes after 24-h Na+-enriched μCi/ml86Rb μCi/ml 22Na 6-h (line 1)- 2)-enriched CS specific ouabain. more details, "Experimental Procedures." Mean values S.E. three experiments performed triplicate (ionic content) quadruplicate (chromatin cleavage) given.a p Open table new tab IIEffect lactate dehydrogenase release serum-deprived heat-treated VSMCChromatin fragmentsLDH releaseControl cells%%1. Serum-supplied cells2.3 0.41.3 0.4ap 0.05 cells.1.1 0.41.2 0.32. Serum-deprived cells4.5 0.71.8 0.3bp cells.2.2 0.51.8 0.43. Serum-supplied, 0.61.6 0.410.2 2.4cp serum-supplied, heat-untreated cells.11.0 2.8cp cells.VSMC growing six-well (lactate (LDH) release) assay) plates preincubated 3 part experiments, subjected 30-min 46 °C (heat-treated 3). They (lines 3) during 8 LDH Procedures" spectrophotometric kit (Sigma), respectively. Means (LDH cells.b cells.c IEffect +-depleted nontransfected adenovirusIncubation mediumChromatin fragmentsVSMCVSMC-MYCVSMC-E1A10% free%%%1. Control1.9 0.36.6 0.92.7 0.515.9 1.54.1 0.428.3 3.62. Ouabain1.6 0.32.5 0.6bp controls.1.4 0.33.1 0.4bp controls.1.5 0.3ap controls.5.7 controls.3. K+depletion1.7 0.42.4 controls.1.6 0.43.5 0.7bp controls.1.9 0.2ap controls.5.8 1.9bp controls.Cells K+-containing consisting 91 NaCl, KCl, 0.9 mmNaH2PO4, 44 NaHCO3, 1.8 CaCl2, 0.8 MgCl2, 33 HEPES, d-glucose 7.4) (equimolar chloride, indicated. VSMC-MYC VSMC-E1A, serum-free medium. After (VSMC-MYC VSMC-E1A), (VSMC-MYC, VSMC-E1A) controls.b controls. Figure 4Effect a, kinetics accumulation (curves 4) (curves3 ng/ml anti-human (mouse monoclonal IgM, Upstate Biotechnology, Lake Placid, NY). b, 0.25 μm 350 mannitol. added given.View Large Image ViewerDownload (PPT)Table IVEffect cellsConcentration mediumIntracellular fragmentsControl cellsNa+iK+iNa+iK+iControl+Fas-LControl+Fas-Lmmnmol/mg 5183 331545 961137 10ap cells.107 cells.4.9 2.128.5 3.316.0 4.6bp 0.01 cells.42.7 5.0bp 128162 331584 99204 30498 474.0 0.620.0 2.25.7 2.0bp cells.22.7 5.3bp cells.To 7 (Fas-L). K+content 86Rb given. VSMC-E1A seeded 75-cm2flasks treated 50 20 EDTA 8.0), SDS, proteinase K. 250 RNase A, 37 °C. phenol-chloroform extraction, precipitated acetate ethanol (1:2, v:v). precipitate water, μg CoCl2, 0.2 dithiothreitol, cacodilate, mm[32P]dCTP (3000 Ci/mmol), units/μl terminal deoxynucleotidyl transferase (final volume, μl). °C, aliquots loaded 1.5% agarose gel, run V 3–4 onto nylon membrane (Hybond N+, Amersham Pharmacia Biotech), analyzed PhosphorImager 21Hamet Richard Teiger Orlov Gaboury Gossard Hypertension. 642-648Crossref (248) 20-cm2flasks scratched, centrifuge tubes, saline, lysed Buffer A Tris-HCl 7.4), EGTA, 0.1% CHAPS. Then, 50–100 600 dithiothreitol 40 YVAD-AMC DEVD-AMC, Caspase-1 inhibitors (Ac-YVAD-CHO Ac-DEVD-CHO, respectively), diluted 15-fold 80 glycine-NaOH 10). Fluorescence samples λex 365 nm λem 465 calibrated AMC range nm. Caspase ∼2 107 ice sedimented. pellet 0.3 way VSMC. Protein Bradford method. exchangeable extra- 86Rb, (22Orlov Membr. 125-135Crossref establish isotopes, 12 (22Na)- (86Rb)-well ∼106 86RbCl μCi/ml22NaCl compositions Tables III IV radioactivity. end time, four times W, MgCl2 mmHEPES-Tris SDS/EDTA mixture. One applied Whatman C filter being 5-ml W negative pressure 20–30 Hg. liquid scintillation analyzer. determined asA/am, where (cpm), (86Rb) (cpm/nmol), m protein method Lowry. Previously, pathways deprivation can be heightened transfection functional analogue adenoviral respectively) these study regulation ratio, inhibited depleting K+. Na+content augmented 38 283 30 nmol/mg protein, K+i dropped 405 41 131 19 (Fig. 1, curves Prolongation up significantly modify Na+i VSMC-E1A. modulation Na+iand observed (data shown). 10-fold lymphocytes curve consistent free Na+iconcentration human peripheral fluorescent Na+-indicator, binding benzofuran isophthalate (23Webb G.D. Taylor Oh V.M.S. Yeo S.-B. Ng L.L. Sci. 88: 695-700Crossref Considering results, effect [Na+]i/[K+]iratio 1–2 inhibition. As seen drastically potentiated 4.5 0.4 26.3 3.0% media, Treatment blocked base-line withdrawal-induced cleavage. Similar also (TableI). procedure sharply (Table I). b shows apart kinase serine-threonine phosphatase. staurosporine acid, increased 30.1 4.7 28.3 4.2%, a), compounds b). mannitol (Fig.2 This previous further confirmed phase microscopy analysis laddering. Fig.3 pretreatment decreased number prevented laddering reduced molecular weight 3′-end-labeled oligonucleosomal (Fig.3 B). underwent acid reported heat stress triggers necrosis but does (24Champagne M.-J. Dumas Bennett 1999; 33: 906-913Crossref (58) Indeed, TableII, (46 min) modulate led 5-fold release, marker necrosis. apoptosis, observe stress-induced release. Viewed collectively, data demonstrate against necrotic death. 7-h monotonously ∼5%. 1.5-h (anti-human Fas, mouse IgM), change much next 5.5 These Pinkosk