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Critical role of the α1-Na+, K+-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain

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Abstract

In rodents, ubiquitous α1-Na+, K+-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~103-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na+, K+-ATPase isoforms, we compared the effects of ouabain on intracellular Na+ and K+ content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 μM ouabain dramatically increased the intracellular [Na+]/[K+] ratio to the same extend as in RASMC and RAEC treated with 3000 μM ouabain. In 24, 3 μM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3000–5000 μM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1R/R mouse, ouabain triggered death of MASMC from α1S/S mouse expressing human α1-Na+, K+-ATPase. Furthermore, transfection of HUVEC with rat α1R-Na+, K+-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na+, K+-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery.

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References

  1. Therien AG, Blostien R (2000) Mechanisms of sodium pump regulation. Am J Physiol 279:C541–C566

    CAS  Google Scholar 

  2. Scheiner-Bobis G (2002) The sodium pump. Its molecular properties and mechanics of ion transport. Eur J Biochem 269:2424–2433

    Article  CAS  PubMed  Google Scholar 

  3. Bagrov AY, Shapiro JI, Fedorova OV (2009) Endogenous cardiotonic steroids: physiology, pharmacology, and novel therapeutic targets. Pharmacol Rev 61:9–38

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Schoner W, Scheiner-Bobis G (2007) Endogenous and exogenous cardiac glycosides: their role in hypertension, salt metabolism, and cell growth. Am J Physiol Cell Physiol 293:C509–C536

    Article  CAS  PubMed  Google Scholar 

  5. Lingrel JB, Argьello JM, Van Huysse JW, Kuntzweiler TA (1997) Cation and cardiac glycoside binding sites of the Na, K-ATPase. Ann N.Y. Acad Sci 843:194–206

    Article  Google Scholar 

  6. Lingrel JB, Williams MT, Vorhees CV, Moseley AE (2007) Na, K-ATPase and the role of a isoforms in behaviour. J Bioenerg Bioeng 39:385–389

    Article  CAS  Google Scholar 

  7. Dostanic-Larson I, Van Huysse JW, Lorenz JN, Lingrel JB (2005) The highly conserved cardiac glycoside binding site of Na, K-ATPase plays a role in blood pressure regulation. Proc Natl Acad Sci USA 102:15845–15850

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Hou X, Theriault SF, Dostanic-Larson I, Moseley AE, Lingrel JB, Wu H, Dean S, Van Huysse JW (2009) Enhanced pressor response to increased CSF sodium concentration and to central angiotensin I in heterozygous a2 Na, K-ATPase knockout mice. Am J Physiol Regul Integr Comp Physiol 295:R1427–R1438

    Article  Google Scholar 

  9. Dostanic I, Lorenz JN, Schultz JEJ, Grupp IL, Neumann JC, Wani MA, Lingrel JB (2003) The α2 isoform of Na, K-ATPase mediates ouabain-induced cardiac inotropy in mice. J Biol Chem 278:53026–53034

    Article  CAS  PubMed  Google Scholar 

  10. Radzyukevich TL, Lingrel JB, Heiny JA (2009) The cardiac glycoside binding site of the Na, K-ATPase α2 isoform plays a role in the dynamic regulation of active transport in skeletal muscle. Proc Natl Acad Sci USA 106:2565–2570

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Loreaux EL, Kaul B, Lorenz JN, Lingrel JB (2008) Ouabain-sensitive α1 Na, K-ATPase enhances natriuretic response to saline load. J Am Soc Nephrol 19:1947–1954

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Ledbetter ML, Young GJ, Wright ER (1986) Cooperation between epithelial cells demonstrated by potassium transfer. Am J Physiol 250:C306–C313

    CAS  PubMed  Google Scholar 

  13. Bolivar JJ, Lazaro A, Fernandez S, Stefani E, Pena-Cruz V, Lechene C, Cereijido M (1987) Rescue of a wild-type MDCK cell by a ouabain-resistant mutant. Am J Physiol 253:C151–C161

    CAS  PubMed  Google Scholar 

  14. Orlov SN, Thorin-Trescases N, Pchejetski D, Taurin S, Farhat N, Tremblay J, Thorin E, Hamet P (2004) Na+/K+ pump and endothelial cell survival: [Na+]i/[K+]i-independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na+]i. Pflug Arch 448:335–345

    Article  CAS  Google Scholar 

  15. Chueh S-C, Guh J-H, Chen J, Lai M-K, Teng C-M (2001) Dual effect of ouabain on the regulation of proliferation and apoptosis in human prostatic smooth muscle cells. J Urol 166:347–353

    Article  CAS  PubMed  Google Scholar 

  16. McConkey DJ, Lin Y, Nutt LK, Ozel HZ, Newman RA (2000) Cardiac glycosides stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic human prostate adenocarcinoma cells. Cancer Res 60:3807–3812

    CAS  PubMed  Google Scholar 

  17. Kurosawa M, Tani Y, Nishimura S, Numazawa S, Yoshida T (2001) Distinct PKC isozymes regulate buffalin-induced diiferentiation and apoptosis in human monocyte cells. Am J Physiol Cell Physiol 280:C459–C464

    CAS  PubMed  Google Scholar 

  18. Perne A, Muellner MK, Steinrueck M, Craig-Mueller N, Mayerhofer J, Schwarzinger I, Sloane M, Uras IZ, Hoermann G, Nijman SMB (2009) Cardiotonic glycosides induce cell death in human cells by inhibiting general protein synthesis. PLoS One 4:e8292

    Article  PubMed Central  PubMed  Google Scholar 

  19. Kulikov A, Eva A, Kirch U, Boldyrev A, Scheiner-Bobis G (2007) Ouabain activates signaling pathways associated with cell death in human neuroblastoma. Biochim Biophys Acta 1768:1691–1702

    Article  CAS  PubMed  Google Scholar 

  20. Hennion JP, el-Masri MA, Huff MO, el-Mailakh RS, (2002) Evaluation of neuroprotection by lithium and valproic acid against ouabain-induced cell damage. Bipolar Disord 4:201–206

    Article  CAS  PubMed  Google Scholar 

  21. Rosen H, Glukmann V, Feldman T, Fridman E, Lichtstein D (2004) Cardiac steroids induce changes in recyling of the plasma membrane in human NT2 cells. Mol Biol Cell 15:1044–1054

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Orlov SN, Thorin-Trescases N, Kotelevtsev SV, Tremblay J, Hamet P (1999) Inversion of the intracellular Na+/K+ ratio blocks apoptosis in vascular smooth muscle at a site upstream of caspase-3. J Biol Chem 274:16545–16552

    Article  CAS  PubMed  Google Scholar 

  23. Akimova OA, Mongin AA, Hamet P, Orlov SN (2006) The rapid decline of MTT reduction is not a marker of death signaling in ouabain-treated cells. Cell Mol Biol 52(8):71–77

    CAS  PubMed  Google Scholar 

  24. Pchejetski D, Taurin S, der Sarkissian S, Lopina OD, Pshezhetsky AV, Tremblay J, DeBlois D, Hamet P, Orlov SN (2003) Inhibition of Na+, K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio. Biochem Biophys Res Commun 301:735–744

    Article  CAS  PubMed  Google Scholar 

  25. Contreras RG, Flores-Maldonado C, Lazaro A, Shoshani L, Flores-Benitez D, Larre I, Cereijido M (2004) Ouabain binding to Na+, K+-ATPase relaxes cell attachment and sends a specific signal (NACos) to the nucleus. J Membr Biol 198:147–158

    Article  CAS  PubMed  Google Scholar 

  26. Akimova OA, Bagrov AY, Lopina OD, Kamernitsky AV, Tremblay J, Hamet P, Orlov SN (2005) Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells. J Biol Chem 280:832–839

    Article  CAS  PubMed  Google Scholar 

  27. Orlov SN, Hamet P (2006) The death of cardiotinic steroid-treated cells: evidence of N+ i, K+ i-independent H+ i-sensitive signaling. Acta Physiol 187:231–240

    Article  CAS  Google Scholar 

  28. Voghel G, Thorin-Trescases N, Farhat N, Nguyen A, Villeneuve L, Mamarbachi AM, Fortier A, Perrault LP, Carrier M, Thorin E (2007) Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress asociated with cardiovascular risk factors. Mech Ageing Dev 128:662–671

    Article  CAS  PubMed  Google Scholar 

  29. Kuo Y-H, Abdullaev IF, Hyzinski-Garcia MC, Mongin AA (2014) Effects of alternative splicing on the function of bestrophin-1 calcium-activated chlroide channels. Biochem J 458:575–583

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Mongin AA, Hyzinski-Garcia MC, Vincent MY, Keller RW (2011) A simple method for measuring intracellular activities of glutamate synthetase and glutaminase in glial cells. Am J Physiol Cell Physiol 301:C814–C822

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Dostanic I, Schultz JEJ, Lorenz JN, Lingrel JB (2004) The α1 isoform of Na,K-ATPase regulates contractility and functionally interacts and co-localizes with the Na/Ca-exchanger in heart. J Biol Chem 279(52):54053–54061

    Article  CAS  PubMed  Google Scholar 

  32. Wansapura AN, Lasko VM, Lingrel JB, Lorenz JN (2011) Mice expressing ouabain-sensitive a1-Na, K-ATPase have increased susceptibility to pressure overload-induced cardiac hypertrtophy. Am J Physiol Heart Circ Physiol 300:H347–H355

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Ray JL, Leach R, Herbert JM, Benson M (2002) Isolation of vascular smooth muscle cells from a sngle murine aorta. Methods Cell Sci 23:185–188

    Google Scholar 

  34. Orlov SN, Thorin-Trescases N, Dulin NO, Dam T-V, Fortuno MA, Tremblay J, Hamet P (1999) Activation of cAMP signaling transiently inhibits apoptosis in vascular smooth muscle cells in a site upstream of caspase 3. Cell Death Differ 6:661–672

    Article  CAS  PubMed  Google Scholar 

  35. Jewell EA, Lingrel JB (1991) Comparison of the substrate dependence properties of the rat Na, K, ATPase α1, α2, and α3 isoforms expressed in HeLa cells. J Biol Chem 266:16925–16930

    CAS  PubMed  Google Scholar 

  36. Akimova OA, Tremblay J, Van Huysse JW, Hamet P, Orlov SN (2010) Cardiotonic steroid-resistant α1-Na+, K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Na+ i, K+ i-independent mechanism. Apoptosis 15:55–62

    Article  CAS  PubMed  Google Scholar 

  37. Thornton TM, Rincon M (2009) Non-classical p38 MAP kinase functions: cell cycle checkpoints and survival. Int J Biol Sci 5:44–51

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Boutros T, Chevet E, Metrakos P (2008) Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase regulation: roles in cell growth, death, and cancer. Pharmacol Rev 60:261–310

    Article  CAS  PubMed  Google Scholar 

  39. Kim EK, Choi EJ (2010) Pathological toles of MAPK signaling pathways in human diseases. Biochim Biophys Acta 1802:396–405

    Article  CAS  PubMed  Google Scholar 

  40. Munshi A, Ramesh R (2013) Mitogen-activated protein-kinases and their role in radiation response. Genes Cancer 4:401–408

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Li Q, Chen M, Liu H, Yang L, Yang T, He G (2014) The dual role of ERK signaling in the apoptotosis of nerones. Front Biosci 19:1411–1417

    Article  Google Scholar 

  42. Akimova OA, Lopina OD, Rubtsov AM, Gekle M, Tremblay J, Hamet P, Orlov SN (2009) Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na+i/K+i-independent signaling. Apoptosis 14:1266–1273

    Article  CAS  PubMed  Google Scholar 

  43. Akimova OA, Lopina OD, Rubtsov AM, Hamet P, Orlov SN (2010) Investigation of mechanism of activation of p38 MAPK by cardiotonic steroids in epithelial cells from distal tubules. Biochemistry 75:1070–1078

    Google Scholar 

  44. Pierre S, Compe E, Grillasca JP, Plannells R, Sampol J, Pressley TA, Maixent JM (2001) RP-PCR detection of Na, K-ATPase subunit isoforms in human unbilical vein endothelial cells (HUVEC): evidence for the presence of α1 and β3. Cell Mol Biol 47:319–324

    CAS  PubMed  Google Scholar 

  45. Hansen O (2004) Quantification of α-subunit isoforms of Na-K-ATPase in rat resistant vessels. Acta Physiol Scand 180:49–56

    Article  CAS  PubMed  Google Scholar 

  46. Golovina VA, Song H, James PF, Lingrel JB, Blaustein MP (2003) Na+ pump α2-subunit expression modulates Ca2+ signaling. Am J Physiol 284:C475–C486

    Article  CAS  Google Scholar 

  47. Song H, Thompson SM, Blaustein MP (2013) Nanomolar ouabain augments Ca2+-signalling in rat hippocampal nerones and glia. J Physiol 591:1671–1689

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Benarrroch EE (2011) Na+, K+-ATPase: functions in the nervous system and involvement in the neurologic disease. Neurology 76:287–293

    Article  Google Scholar 

  49. Mobasheri A, Avila J, Cozar-Castellano I, Brownleader MD, Trevan M, Francis MJO, Lamb JF, Martin-Vassalo P (2000) Na+, K+-ATPase isozyme diversity: comparative biochemistry and physiological implications of novel functional interactions. Biosci Rep 20:51–91

    Article  CAS  PubMed  Google Scholar 

  50. Lingrel JB (2010) The physiological significance of the cardiotonic steroid/ouabain-binding site of the Na, K-ATPase. Annu Rev Physiol 72:395–412

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. Xiao AY, Wei L, Xia S, Rothman S, Yu SP (2002) Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurones. J Neurosci 22:1350–1362

    CAS  PubMed  Google Scholar 

  52. Liu J, Xie Z (2010) The sodium pump and cardiotonic steroids-induced signal transduction protein kinases and calcium-signaling microdomain in regulation of transporter traficking. Biochim Biophys Acta 1802:1237–1245

    Article  CAS  PubMed  Google Scholar 

  53. Lauf PK, Alqahtani T, Flues K, Meller J, Adragna NC (2015) Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak. Am J Physiol Cell Physiol 308:C51–C60

    Article  CAS  PubMed  Google Scholar 

  54. Koltsova SV, Trushina Y, Haloui M, Akimova OA, Tremblay J, Hamet P, Orlov SN (2012) Ubiquitous [Na+]i/[K+]i-sensitive transcriptome in mammalian cells: evidence for Ca2+ i-independent excitation-transcription coupling. PLoS One 7:e38032

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  55. Orlov SN, Hamet P (2015) Salt and gene expression: evidence for Na+i, K+i-mediated signaling pathways. Pflug Arch Eur J Physiol 467:475–487

    Article  Google Scholar 

  56. Goldin AC, Safa AR (2015) Digitalis and cancer. Lancet1134

  57. Haux J, Kiepp O, Spigset O, Tretli S (2001) Digitoxin medication and cancer: case control and inernal dose response studies. BMC Cancer 1:11

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  58. Platz EA, Yegnasubramanian S, Liu JO, Chong CR, Shim JS, Kenfield SA, Stampfler MJ, Willett WC, Giovannucci E, Nelson WG (2011) A novel two-stage, transdisciplinary study identifies digoxin as a possible drug for prostate cancer treatment. Cancer Discov 1:68–77

    Article  CAS  PubMed  Google Scholar 

  59. Newman RA, Yang P, Pawlus AD, Block KI (2008) Cardiac glycosides as novel cancer therapeutic agents. Mol Interv 8:36–49

    Article  CAS  PubMed  Google Scholar 

  60. Mijatovic T, Van Quaquebeke E, Delest B, Debeir O, Darro F, Kiss R (2007) Cardiotonic steroids on the road to anti-cancer therapy. Biochim Biophys Acta 1776:32–57

    CAS  PubMed  Google Scholar 

  61. Weidemann H (2005) Na/K-ATPase, endogenous digitalis-like compounds and cancer development—a hypothesis. Front Biosci 10:2165–2176

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was supported in part by Grants from the Russian Foundation for Fundamental Research 09-0073/04, 14-04-31705 and 15-04-00101 (to S.N.O. and A.O.A.), grant from the Russian Scientific Foundation 14-15-00006 (to A.O.A. and S.N.O.), grant from the National Institutes of Health (NS061953 to A.A.M.) and Predoctoral Fellowship from American Heart Association 14PRE18360017 to J.L.

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Authors and Affiliations

  1. Laboratory of Biological Membranes, Faculty of Biology, M. V. Lomonosov Moscow State University, room 169, Vorob’evy Gory 1/12, Moscow, 119899, Russia

    Olga A. Akimova, Artem M. Tverskoi, Larisa V. Smolyaninova, Olga D. Lopina & Sergei N. Orlov

  2. Albany Medical College, Albany, NY, USA

    Alexander A. Mongin

  3. University of Chicago, Chicago, IL, USA

    Jennifer La & Nickolai O. Dulin

  4. Tomsk State University, Tomsk, Russia

    Sergei N. Orlov

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  1. Olga A. Akimova
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  2. Artem M. Tverskoi
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  3. Larisa V. Smolyaninova
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  4. Alexander A. Mongin
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  5. Olga D. Lopina
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  6. Jennifer La
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  8. Sergei N. Orlov
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Correspondence to Sergei N. Orlov.

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Akimova, O.A., Tverskoi, A.M., Smolyaninova, L.V. et al. Critical role of the α1-Na+, K+-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain. Apoptosis 20, 1200–1210 (2015). https://doi.org/10.1007/s10495-015-1144-y

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