Abstract
Long-term study on the identification of Na,K-ATPase endogenous inhibitors in mammalian tissues has resulted in the discovery of ouabain, marinobufagenin (MBG), and other cardiotonic steroids (CTS) in the blood plasma. Production of ouabain and MBG is increased in essential hypertension and other diseases associated with hypervolemia. Here, we compared the effects of ouabain and MBG on the Na,K-ATPase activity (measured as the transport of Na+, K+, and Rb+ ions) and proliferation and death of human renal epithelial cells (HRECs) and human umbilical vein endothelial cells (HUVEC) expressing α1-Na,K-ATPase. Ouabain concentration that provided the half-maximal inhibition of the Rb+ influx (IC50) into HRECs and HUVECs was 0.07 μM. In both types of cells, the IC50 values for MBG were 10 times higher than for ouabain. Incubation of HREC and HUVEC with 0.001-0.01 μM ouabain for 30 h resulted in 40% increase in the [3H]thymidine incorporation into DNA; further elevation of ouabain concentration to 0.1 μM completely suppressed DNA synthesis. MBG at the concentration of 0.1 μM activated DNA synthesis by 25% in HRECs, but not in HUVECs; 1 μM MBG completely inhibited DNA synthesis in HRECs and by 50% in HUVECs. In contrast to HRECs, incubation of HUVECs in the serum-free medium induced apoptosis, which was almost completely suppressed by ouabain and MBG at the concentrations of 0.1 and 3 μM, respectively. Based on these data, we can conclude that (i) the effect of MBG at the concentrations detected in the blood plasma (<0.01 μM) on HRECs and HUVECs was not due to the changes in the [Na+]i/[K+]i ratio; (ii) the effect of physiological concentrations of ouabain on these cells might be mediated by the activation of Na,K-ATPase, leading to cell proliferation.
Similar content being viewed by others
Abbreviations
- Ac-DEVD-AMC:
-
N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin
- CTS:
-
cardiotonic steroid
- HREC:
-
human renal epithelial cell
- HUVEC:
-
human umbilical vein endothelial cell
- MBG:
-
marinobufagenin
- PAEC:
-
porcine aorta endothelial cell
- REC:
-
renal epithelial cel
REFERENCES
Krikler, D. M. (1985) The foxglove, “the old woman from Shropshire” and William Withering, J. Am. Coll. Cardiol., 5, 3A-9A, doi: 10.1016/s0735-1097(85)80457-5.
Krenn, L., and Kopp, B. (1998) Bufadienolides from animal and plant sources, Phytochemistry, 48, 1-29, doi: 10.1016/s0031-9422(97)00426-3.
Schatzmann, H. J. (1953) Cardiac glycosides as inhibitors of active potassium and sodium transport by erythrocyte membrane, Helv. Physiol. Pharmacol. Acta, 11, 346-354.
Skou, J. Chr. (1960) Further investigations on a Mg++ + Na+-activated adenosine triphosphatase, possibly related to the active, linked transport of Na+ and K+ across the nerve membrane, Biochim. Biophys. Acta, 42, 6-23, doi: 10.1016/0006-3002(60)90746-0.
Skou, J. Chr. (1957) The influence of some cations on an adenosine triphosphatase from peripheral nerves, Biochim. Biophys. Acta, 23, 394-401.
Szent-Gyorgyi, A. (1953) Chemical physiology of contraction in body and heart muscle, Science, 119, 803, doi: 10.1126/science.119.3101.803.
Khalaf, F. K., Dube, P., Mohamed, A., Tian, J., Malhotra, D., Haller, S. T., and Kennedy, D. J. (2018) Cardiotonic steroids and the sodium trade balance: new insights into trade-off mechanisms mediated by the Na+/K+-ATPase, Int. J. Mol. Sci., 19, 2576, doi: 10.3390/ijms19092576.
Blanco, G., and Mercer, R. W. (1998) Isozymes of the Na,K-ATPase: heterogeneity in structure, diversity in function, Am. J. Physiol. Ren. Physiol., 275, F633-F650, doi: 10.1152/ajprenal.1998.275.5.F633.
Therien, A. G., and Blostein, R. (2000) Mechanisms of sodium pump regulation, Am. J. Physiol. Cell Physiol., 279, C541-C566, doi: 10.1152/ajpcell.2000.279.3.C541.
Rajasekaran, S. A., Gopal, J., Willis, D., Espineda, C., Twiss, J. L., and Rajasekaran, A. K. (2004) Na,K-ATPase β1-subunit increases the translation efficiency of the α1-subunit in MSV-MDCK cells, Mol. Biol. Cell, 15, 3224-3232, doi: 10.1091/mbc.E04-03-0222.
Herrera, V. L., Emanuel, J. R., Ruiz-Opazo, N., Levenson, R., and Nadal-Ginard, B. (1987) Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications, J. Cell Biol., 105, 1855-1865, doi: 10.1083/jcb.105.4.1855.
Dmitrieva, R. I., and Doris, P. A. (2003) Ouabain is a potent promoter of growth and activator of ERK1/2 in ouabain-resistant rat renal epithelial cells, J. Biol. Chem., 278, 28160-28166, doi: 10.1074/jbc.M303768200.
Pierre, S., Compe, E., Grillasca, J. P., Plannells, R., Sampol, J., Pressley, T. A., and Maixent, J. M. (2001) RT-PCR detection of Na,K-ATPase subunit isoforms in human umbilical vein endothelial cells (HUVEC): evidence for the presence of alpha1 and beta3, Cell. Mol. Biol., 47, 319-324.
Zahler, R., Sun, W., Ardito, T., and Kashgarian, M. (1996) Na-K-ATPase alpha-isoform expression in heart and vascular endothelia: cellular and developmental regulation, Am. J. Physiol. Cell Physiol., 270, C361-C371, doi: 10.1152/ajpcell.1996.270.1.C361.
Orlov, S. N., Klimanova, E. A., Tverskoi, A. M., Vladychenskaya, E. A., Smolyaninova, L. V., and Lopina, O. D. (2017) Na+i,K+i-dependent and -independent signaling triggered by cardiotonic steroids: facts and artifacts, Mol. Basel Switz., 22, 635, doi: 10.3390/molecules22040635.
Riganti, C., Campia, I., Kopecka, J., Gazzano, E., Doublier, S., Aldieri, E., Bosia, A., and Ghigo, D. (2011) Pleiotropic effects of cardioactive glycosides, Curr. Med. Chem., 18, 872-885, doi: 10.2174/092986711794927685.
Dvela, M., Rosen, H., Feldmann, T., Nesher, M., and Lichtstein, D. (2007) Diverse biological responses to different cardiotonic steroids, Pathophysiology, 14, 159-166, doi: 10.1016/j.pathophys.2007.09.011.
Katz, A., Lifshitz, Y., Bab-Dinitz, E., Kapri-Pardes, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (2010) Selectivity of digitalis glycosides for isoforms of human Na,K-ATPase, J. Biol. Chem., 285, 19582-19592, doi: 10.1074/jbc.M110.119248.
Tverskoy, A. M., Lokteva, V. A., Orlov, S. N., and Lopina, O. D. (2019) Change in the conformation of α1-Na,K-ATPase resistant and sensitive to cardiotonic steroids upon binding of ouabain, digoxin and marinobufagenin, Receptors and Intracellular Signaling (collection of articles), 1, 181-185.
Reich, H., Tritchler, D., Herzenberg, A. M., Kassiri, Z., Zhou, X., Gao, W., and Scholey, J. W. (2005) Albumin activates ERK via EGF receptor in human renal epithelial cells, J. Am. Soc. Nephrol., 16, 1266-1278, doi: 10.1681/ASN.2004030222.
Hartree, E. F. (1972) Determination of protein: a modification of the Lowry method that gives a linear photometric response, Anal. Biochem., 48, 422-427, doi: 10.1016/0003-2697(72)90094-2.
Orlov, S. N., Thorin-Trescases, N., Kotelevtsev, S. V., Tremblay, J., and 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, doi: 10.1074/jbc.274.23.16545.
Pchejetski, D., Taurin, S., Der Sarkissian, S., Lopina, O. D., Pshezhetsky, A. V., Tremblay, J., deBlois, D., Hamet, P., and Orlov, S. N. (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, doi: 10.1016/s0006-291x(02)03002-4.
Shiyan, A. A., Sidorenko, S. V., Fedorov, D., Klimanova, E. A., Smolyaninova, L. V., Kapilevich, L. V., Grygorczyk, R., and Orlov, S. N. (2019) Elevation of intracellular Na+ contributes to expression of early response genes triggered by endothelial cell shrinkage, Cell. Physiol. Biochem., 53, 638-647, doi: 10.33594/000000162.
Wickham, H. (2009) Ggplot2: Elegant Graphics for Data Analysis, 2nd Edn., Springer Publishing Company.
Akimova, O. A., Hamet, P., and Orlov, S. N. (2007) [Na+]i/[K+]i-independent death of ouabain-treated renal epithelial cells is not mediated by Na+,K+-ATPase internalization and de novo gene expression, Pflügers Arch., 455, 711-719, doi: 10.1007/s00424-007-0283-6.
Palmer, R. F., Lasseter, K. C., and Melvin, S. L. (1966) Stimulation of Na+ and K+ dependent adenosine triphosphatase by ouabain, Arch. Biochem. Biophys., 113, 629-633, doi: 10.1016/0003-9861(66)90240-2.
Godfraind, T., and Ghysel-Burton, J. (1977) Binding sites related to ouabain-induced stimulation or inhibition of the sodium pump, Nature, 265, 165, doi: 10.1038/265165a0.
Klimanova, E. A., Petrushanko, I. Y., Mitkevich, V. A., Anashkina, A. A., Orlov, S. N., Makarov, A. A., and Lopina, O. D. (2015) Binding of ouabain and marinobufagenin leads to different structural changes in Na,K-ATPase and depends on the enzyme conformation, FEBS Lett., 589, 2668-2674, doi: 10.1016/j.febslet.2015.08.011.
Aydemir-Koksoy, A., Abramowitz, J., and Allen, J. C. (2001) Ouabain-induced signaling and vascular smooth muscle cell proliferation, J. Biol. Chem., 276, 46605-46611, doi: 10.1074/jbc.M106178200.
Abramowitz, J., Dai, C., Hirschi, K. K., Dmitrieva, R. I., Doris, P. A., Liu, L., and Allen, J. C. (2003) Ouabain- and marinobufagenin-induced proliferation of human umbilical vein smooth muscle cells and a rat vascular smooth muscle cell line, A7r5, Circulation, 108, 3048-3053, doi: 10.1161/01.CIR.0000101919.00548.86.
Li, M., Wang, Q., and Guan, L. (2007) Effects of ouabain on proliferation, intracellular free calcium and c-myc mRNA expression in vascular smooth muscle cells, J. Comp. Physiol. B, 177, 589-595, doi: 10.1007/s00360-007-0157-4.
Chueh, S.-C., Guh, J.-H., Chen, J., Lai, M.-K., and Teng, C.-M. (2001) Dual effects of ouabain on the regulation of proliferation and apoptosis in human prostatic smooth muscle cells, J. Urol., 166, 347-353, doi: 10.1016/S0022-5347(05)66157-5.
Khundmiri, S. J., Metzler, M. A., Ameen, M., Amin, V., Rane, M. J., and Delamere, N. A. (2006) Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells, Am. J. Physiol. Cell Physiol., 291, C1247-C1257, doi: 10.1152/ajpcell.00593.2005.
Saunders, R., and Scheiner-Bobis, G. (2004) Ouabain stimulates endothelin release and expression in human endothelial cells without inhibiting the sodium pump, Eur. J. Biochem., 271, 1054-1062, doi: 10.1111/j.1432-1033.2004.04012.x.
Qiu, J., Gao, H.-Q., Zhou, R.-H., Liang, Y., Zhang, X.-H., Wang, X.-P., You, B.-A., and Cheng, M. (2007) Proteomics analysis of the proliferative effect of low-dose ouabain on human endothelial cells, Biol. Pharm. Bull., 30, 247-253, doi: 10.1248/bpb.30.247.
Tverskoi, A. M., Sidorenko, S. V., Klimanova, E. A., Akimova, O. A., Smolyaninova, L. V., Lopina, O. D., and Orlov, S. N. (2016) Effects of ouabain on proliferation of human endothelial cells correlate with Na+,K+-ATPase activity and intracellular ratio of Na+ and K+, Biochemistry (Moscow), 81, 876-883, doi: 10.1134/S0006297916080083.
Bolivar, J. J., Lazaro, A., Fernandez, S., Stefani, E., Pena-Cruz, V., Lechene, C., and Cereijido, M. (1987) Rescue of a wild-type MDCK cell by a ouabain-resistant mutant, Am. J. Physiol. Cell Physiol., 253, C151-C161, doi: 10.1152/ajpcell.1987.253.1.C151.
Orlov, S. N., Thorin-Trescases, N., Pchejetski, D., Taurin, S., Farhat, N., Tremblay, J., Thorin, E., and 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, Pflügers Arch., 448, 335-345, doi: 10.1007/s00424-004-1262-9.
Orlov, S. N., and Hamet, P. (2005) Apoptosis vs. oncosis: role of cell volume and intracellular monovalent cations, in Cell Volume and Signaling (Lauf, P. K., and Adragna, N. C., eds.) Springer, pp. 219-233, doi: 10.1007/0-387-23752-6_21.
Trevisi, L., Visentin, B., Cusinato, F., Pighin, I., and Luciani, S. (2004) Antiapoptotic effect of ouabain on human umbilical vein endothelial cells, Biochem. Biophys. Res. Commun., 321, 716-721, doi: 10.1016/j.bbrc.2004.07.027.
Askari, A. (2019) The sodium pump and digitalis drugs: dogmas and fallacies, Pharmacol. Res. Perspect., 7, e00505, doi: 10.1002/prp2.505.
Akimova, O. A., Bagrov, A. Y., Lopina, O. D., Kamernitsky, A. V., Tremblay, J., Hamet, P., and Orlov, S. N. (2005) Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells, J. Biol. Chem., 280, 832-839, doi: 10.1074/jbc.M411011200.
Campia, I., Gazzano, E., Pescarmona, G., Ghigo, D., Bosia, A., and Riganti, C. (2009) Digoxin and ouabain increase the synthesis of cholesterol in human liver cells, Cell. Mol. Life Sci., 66, 1580-1594, doi: 10.1007/s00018-009-9018-5.
Campia, I., Sala, V., Kopecka, J., Leo, C., Mitro, N., Costamagna, C., Caruso, D., Pescarmona, G., Crepaldi, T., Ghigo, D., Bosia, A., and Riganti, C. (2012) Digoxin and ouabain induce the efflux of cholesterol via liver X receptor signaling and the synthesis of ATP in cardiomyocytes, Biochem. J., 447, 301-311, doi: 10.1042/BJ20120200.
Sidorenko, S., Klimanova, E., Milovanova, K., Lopina, O. D., Kapilevich, L. V., Chibalin, A. V., and Orlov, S. N. (2018) Transcriptomic changes in C2C12 myotubes triggered by electrical stimulation: role of Ca2+i-mediated and Ca2+i-independent signaling and elevated [Na+]i/[K+]i-ratio, Cell Calcium, 76, 72-86, doi: 10.1016/j.ceca.2018.09.007.
Klimanova, E. A., Tverskoi, A. M., Koltsova, S. V., Sidorenko, S. V., Lopina, O. D., Tremblay, J., Hamet, P., Kapilevich, L. V., and Orlov, S. N. (2017) Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis, Sci. Rep., 7, 45403, doi: 10.1038/srep45403.
Acknowledgements
We express our deep gratitude to Professor A. Ya. Bagrov (St. Petersburg, Russia) for kindly providing marinobufagenin.
Funding
This work was supported by the Russian Foundation for Basic Research (project No. 18-34-00344).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
This article does not contain description of studies with human participants or animals performed by any of the authors. The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Klimanova, E., Fedorov, D., Sidorenko, S. et al. Ouabain and Marinobufagenin: Physiological Effects on Human Epithelial and Endothelial Cells. Biochemistry Moscow 85, 507–515 (2020). https://doi.org/10.1134/S0006297920040112
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0006297920040112