Báo cáo khoa học: No evidence for a role in signal-transduction of Na+/K+-ATPase interaction with putative endogenous ouabain potx

HYPOTHESIS No evidence for a role in signal-transduction of Na + /K + -ATPase interaction with putative endogenous ouabain Otto Hansen Department of Physiology, Aarhus University, DK-8000 A ˚ rhus C, Denmark A cascade of events (signal-transduction), mainly seen in rat cardiac myocytes and renal cells, is thought to occur after ouabain interaction with a minor fraction of Na + /K + - ATPase. A higher intracellular Na + concentration followed sodium pump inhibition by ouabain with a subsequent gradual increase or oscillations in intracellular Ca 2+ con- centration. Whether this increase in intracellular Ca 2+ concentration is part of the cascade, a result of the cascade or a totally independent phenomenon are conflicting interpre- tations that are discussed. At best, however, the cascade is initiated by ouabain concentrations several orders of mag- nitude higher than the measured plasma concentrations of putative endogenous ouabain. The experimentally high ouabain concentration may be critical for another reason. Most tissues contain various isoforms of the catalytic a-peptide of Na + /K + -ATPase with an individual sublocali- zation and, in rats, with different ouabain-sensitivity. The almost ouabain-insensitive a 1 -isoform of Na + /K + -ATPase is essentially unaffected by the high ouabain concentration, whereas ouabain-sensitive a-isoforms, possibly confined to membrane structures near cytosolic microdomains and Na + /Ca 2+ exchangers, may be totally blocked. Classifying endogenous ouabain as a physiological inducer of the sign- aling system on this background seems hazardous. Keywords: a isoforms; endogenous ouabain; Na + /K + - ATPase; ouabain-like factors; OLF; ouabain-insensitive; signal-transduction; sodium pumps. Introduction Ouabain and other cardiac glycosides have been known as specific inhibitors of the sodium pump and the purified Na + /K + -ATPase ever since the very first observation of inhibition of the sodium pump of red cells [1] and inhibition of the isolated Na + /K + -ATPase from crab nerves [2]. Ouabain inhibition of both phenomena was in fact one of the important criteria for the identity between the sodium pump and the purified, though still membrane-embedded Na + /K + -ATPase [3]. Before the inhibitory mechanism had been appreciated, the pharmacological action of cardiac glycosides in congestive heart failure had been known for centuries. The amazing interaction of a plant-derived drug with a membrane receptor of animal cells led to speculations of endogenous mammalian digitalis-like factors (EDLF, reviewed in [4]). The occurence of a ouabain isomer (often called ouabain-like factor, OLF) or even ouabain itself in normal plasma seemed to support these claims [5]. Goto et al. [4] pointed out that a number of criteria should be fulfilled for substances to be considered as candidates for EDLF. Inhibition of Na + /K + -ATPase, as exhibited by, e.g., unsaturated fatty acids [6] and lyso- phospholipids, does not suffice. Neither does EDLF immunoreactivity, as many steroids, lipids and bile acids cross-react with antibodies to cardiac glycosides [4]. Hansen [7] emphasized the vast pool of Na + /K + -ATPase in skeletal muscles, and thus receptors for EDLF/OLF, compared to the measured low secretion rate of OLF from the adreno- cortical gland. A novel role of ouabain in signal-transduction Recently, however, a novel physiological role was attributed to ouabain and thus to endogenous ouabain in addition to the inhibition of Na + /K + -ATPase traditionally associated with the pharmacological action of cardiac glycosides. Based mainly upon experiments with renal cells [8] and ventricular myocytes [9–11], in both cases isolated from the rat, ouabain was suggested to have a hormonal role in signal transduction in addition to the traditional role of Ca 2+ accumulation due to pump inhibition, a reduced Na + gradient and reduced Na + /Ca 2+ exchange resulting in a positive inotropic effect in susceptible tissues (for references see [12]). Generation of the cascade of secondary events was associated with transient oscillations [8] or a more gradual, in some cases independent [10] increase in intracellular Ca 2+ concentration [9–11]. The original observations were accompanied by an enthusiastic commentary in Nature Medicine [13] and a minireview in Eur. J. Biochem. [14]. These authors [13,14] added a more comprehensive and Correspondence to Otto Hansen, Department of Physiology, Aarhus University, Ole Worms Alle ´ 160, DK-8000 A ˚ rhus C, Denmark. Fax: + 45 86129065, Tel.: + 45 89422806, E-mail: oh@fi.au.dk Abbreviations: EDLF, endogenous digitalis-like factor; ER, endo- plasmic reticulum; MAPK, mitogen-activated protein kinase; NF-jB, nuclear factor kappa B; OLF, ouabain-like factor, a ouabain-like isomer; ROS, reactive oxygen species; SR, sarcoplasmic reticulum. (Received 16 January 2003, revised 26 February 2003, accepted 4 March 2003) Eur. J. Biochem. 270, 1916–1919 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03554.x hypothetical cascade of events initiated by ouabain occu- pancy of a small fraction of the sodium pumps. One of them had advocated the role of EDLF for years [15]. Possible pitfalls for a novel role of cardiac glycosides It is generally accepted that the sodium pump and the external aspect of the purified, though still membrane- embedded, Na + /K + -ATPase is the specific receptor for cardiac glycosides, that the binding can be described as a simple bimolecular (second order) reaction with a stoichio- metry of 1 : 1 [12] and that identified amino acid residues of the external loops of the hydrolytic a-peptide are involved in ouabain sensitivity [16]. Caution is needed, however, before classifying OLF or even ouabain as normal steroid hormones functioning in signal-transduction pathways under physiological conditions. The described individual steps [8–11], that may be part of a cascade of events, are not questioned under the conditions of the experiments but serious flaws and failures may invalidate the interpretation and the supposed physiological implication [13,14]. First is the questionable existence of OLF that at best is at an insignificantly low plasma concentration compared to the huge pool of sodium pumps in the mammalian body. Of equal importance is the often ignored fact, that the rat, contrary to most other mammalian species, contains an almost ouabain-insensitive isoform of Na + /K + -ATPase. In most tissues, the Na + /K + -ATPase is a heterodimer consisting of a 112 kDa hydrolytic a-peptide, through which the vectorial transport of Na + and K + takes place, and the small a-glycoprotein (35 kDa for the protein) with chaperone-like function. Four isoforms of the a-peptide have been described (one in testes). In most mammalian species, Na + /K + -ATPase, irrespective of isoform compo- sition, has a very high affinity to the specific inhibitor, ouabain. This is true except for a few rodent species (mainly the rat) in which pumps containing the a 1 -isoform have an extremely low affinity for cardiac glycosides. Most rat tissues moreover have a mixture of ouabain-insensitive and ouabain-sensitive isoforms, of which the latter may have a sublocalization critical for the observations looking at ouabain as a signal-transducer. Experiments pointing to a pivotal role of ouabain in signal-transduction In the following, more details are discussed concerning the experiments with ouabain that led to the explicit formula- tion of a cascade of events. Based mainly on observations with rat myocytes, additional roles in signal-transducing function of ouabain’s interaction with Na + /K + -ATPase were attributed to tyrosine protein kinase, Src, epidermal growth factor receptor and the Ras/MAPK (mitogen- activated protein kinase)-dependent cascade with NF-jB activation and generation of reactive oxygen species (ROS) [9–11]. In each case, the cascade is hypothesized to be elicited by the interaction of a minor fraction of ouabain- altered Na + /K + -ATPase with neighbouring proteins (Src, growth factor receptors and Ras), i.e. by protein–protein interaction. In myocytes, the events from signal-transduc- tion were interpreted as independent of a small increase in [Na + ] i and also occasionally independent of the subsequent increase in [Ca 2+ ] i [10], whereas in other publications, activation of the Ras/MAPK cascade was interpreted as a prerequisite for the increase in [Ca 2+ ] i [11]. Nevertheless, simple inhibition of pump function, by lowering the extracellular K + concentration, was able to mimic ouabain with respect to the cascade in rat cardiac myocytes [9]. In rat renal cells [8] on the other hand, ouabain interaction with a minor fraction of the Na + /K + -ATPase gave rise to the release of Ca 2+ from endoplasmic reticulum (ER) and Ca 2+ influx via voltage-gated and calcium release- activated calcium channels in the plasma membrane. The resulting slow-vawe [Ca 2+ ] i oscillations in turn were inter- preted as activating the transcription factor, NF-jBthat, after translocation to the nucleus, interacted with target genes initiating growth and differentiation. Lowering of the extracellular [K + ] did not mimic ouabain in renal epithelial cells as [Ca 2+ ] i did not change with low extracellular [K + ], though an increase in [Na + ] i was seen [8]. A possible explanation in the latter case could be that pump inhibition is counteracted by hyperpolarization rather than membrane depolarization due to the increased K + gradient according to the Goldman constant-field equation. The Na + /K + - ATPase is indeed electrogenic, contributing in the order of 10 mV to the membrane potential due to the stoichiometry of (3 : 2) Na + /K + -exchange by the pump. On the other hand, according to the pump-leak model, the membrane potential is not established exclusively by the pump. The mentioned, often contradictory observations and interpretations do not add to the credibility of the hypothesis. The main objections to a normal signal- transducing function of ouabain interaction with Na + /K + - ATPase, however, are the following. To place this pheno- menon into a normal physiological framework, the authors [8–11] (a) more or less ignore the presence of a-peptide isoforms of Na + /K + -ATPase with different ouabain affinities in rats; (b) ignore the extremely high concentra- tions of ouabain applied and (c) assume a similar role by putative endogenous ouabain or OLF. Ouabain-sensitive and -insensitive a-isoforms of Na + /K + -ATPase in rats All isoforms of the catalytic a-peptide of Na + /K + -ATPase have similar, high affinity for ouabain [17] with K d in the order of one or a few nanomolar [12,17]. An exception from this general rule is the almost ouabain-insensitive a 1 -isoform seen in rats ([for references, see [18]). In most tissues in the rat, however, this a 1 -isoform, as well as the highly ouabain- sensitive a 2 - (skeletal and heart muscle) and a 3 -isoform (nerve tissue and brain) are present. The exception is the kidney where more than 99.9% of the enzyme consists of the ouabain-insensitive a 1 -isoform [19]. This explains why large concentrations of ouabain (50–500 l M ) are needed to obtain Ca 2+ -oscillations in a reasonable fraction of the rat kidney cells [8], though oscillations were seen in a few cells exposed to 10–100 n M ouabain for 3 h. As ouabain binding to Na + /K + -ATPase is a simple second order reaction [12], 3 h incubation at 37 °C does not seem reasonable for obtaining equilibrium as demonstrated by the statement that Ô…ouabain binding…is expected to increase as a function of timeÕ [8]. However, irrespective of the lower or higher concentration range employed, they are both far Ó FEBS 2003 Endogenous ouabain in normal signal-transduction? (Eur. J. Biochem. 270) 1917 beyond the reported plasma concentration of the putative steroid hormone ouabain or OLF (Fig. 1). In this context, we are even ignoring the fact that Na + /K + -ATPase containing the ouabain-sensitive a 2 -anda 3 -isoforms else- where in the rat would have been totally blocked at micromolar or sub micromolar concentrations of ouabain. Plasma concentrations of putative endogenous ouabain In another recent minireview in Eur. J. Biochem.[20]. endogenous ouabain was claimed to be a novel steroid hormone and the adrenal gland the major place of synthesis of ouabain. However, it is still much debated whether ouabain or OLF really exist in mammalian blood and tissues, and if they do, whether low level production from, e.g. the adrenal glands would suffice [7,21]. In the original publication by Hamlyn et al.[5]onOLFÔ…indistinguish- able from the cardenolide ouabain…Õ they stated an immu- noreactive plasma concentration of 80 ± 18 pmolÆL )1 in normal rats as measured by ELISA. OLF were quantified in plasma from two strains of rat by another group in collaboration with Hamlyn [21] by means of two independ- ent assays, a radioimmunoassay using an anti-ouabain Ig and an enzymatic assay using ouabain-sensitive Na + /K + - ATPase from dog kidney. The concentration range of OLF found in plasma was 25–27 pmolÆL )1 in normotensive and 68–76 pmolÆL )1 in Milan hypertensive rats, i.e., concentra- tions almost 3–6 orders of magnitude lower than employed in experiments focusing on signal-transduction [8–11], see Fig. 1. Higher values based solely on immunoassays have been reported [15] (e.g., during volume expansion in dogs or strenous exercise in man, though still much lower than the concentrations used for signal-transduction) while others have been unable to detect immunoreactive OLF in plasma, adrenal glands and tissues of man at all [22]. Endogenous ouabain was a theme at the 10th International Conference on Na,K-ATPase and Related Cation Pumps in Elsinore, Denmark, in August 2002. The meeting of 250 specialists on Na + /K + -ATPase and related topics didn’t lend much support to the existence of endogenous ouabain and OLF. Assigning ouabain a novel physiological role given this background seems hazardous. Distribution of a-isoforms of Na + /K + -ATPase As to the observations at 100 l M ouabain with rat ventricular myocytes (100 n M with HeLa cells) containing a 1 -aswellasa 2 -isoforms of Na + /K + -ATPase the explan- ation for the apparent signal-tranduction may differ [9–11]. In one of the papers [9] it is mentioned that the rat a 1 -isoform is less sensitive to ouabain but not the significant content of ouabain-sensitive a 2 in rat cardiac muscle [23–25]. The ratio between the a 1 -containing insensitive and a 2 /a 3 -containing sensitive Na + /K + -ATPase in rat cardiac tissue is not well known as only the latter can be determined in [ 3 H]ouabain binding studies. In a membrane fraction from cardiac ventricles of adult rats, high-affinity Na + /K + - ATPase containing a 2 seemed to represent 26% of total activity [25]. A similar distribution could be estimated by using isoform-specific antibodies and Western blots to which homogenates of rat cardiac ventricles had been applied [26]. The a 2 -population of pumps should be totally blocked by the 100 l M ouabain used in experiments with rat ventricular myocytes [9–11]. Due to the preferential locali- zation of the ouabain-sensitive pumps to membrane struc- tures overlying junctional SR/ER [27], an alternative explanation to an apparent signal-transduction role could be a local rise in [Na + ] i resulting in reduced Na + /Ca 2+ - exchange locally. Cellular compartments with larger chan- ges in intracellular Na + and Ca 2+ may thus be the result of a ouabain interaction with subpopulations of Na + /K + - ATPase [27]. An overall increase in intracellular Na + was seen in the experiments with low concentration of extracel- lular [K + ] [8,9], that also mimicked ouabain with respect to the cascade in cardiac myocytes [9]. Other implications of signal-transduction by ouabain Finally, I would like to discuss that a great many studies on rat wild-type, as well as mutant Na + /K + -ATPase, have been carried out with mammalian cell lines transfected with cDNA constructs encoding a ouabain-resistant a 1 -isoform or derived ouabain-resistant a 2 -ora 3 -isoforms [28]. The endogenous ouabain-sensitive Na + /K + -ATPase is knocked-out by incubation of the cells in micromolar concentrations of ouabain [16]. Were signal-transduction of significance in this situation, the phenomenon should be expected in those cells and the results interpreted accordingly. Fig. 1. Ouabain affinities to rat Na + /K + -ATPase isoforms and actual ouabain or OLF concentrations. Thefiguresummarizesthehugevari- ation in ouabain affinities of Na + /K + -ATPase depending on species and a-isoforms, actual concentrations of OLF in rat plasma, actual concentrations of ouabain employed in experiments with renal cells and ventricular myocytes of rats, and the Na + /K + -pump density in rat skeletal muscle. 1918 O. Hansen (Eur. J. Biochem. 270) Ó FEBS 2003 Conclusions Giving ouabain a novel physiological role in signal-trans- duction appears erroneous unless putative endogenous ouabain is produced and present in plasma at reasonable concentrations compared to the affinities with Na + /K + - ATPase. In both respects, convincing data fulfilling a number of criteria for EDLF/OLF are unavailable. More- over, experiments on rat tissue are unfeasible for the demonstration of signal-transduction as ouabain-sensitive a-isoforms of Na + /K + -ATPase, possibly confined to membrane structures near cytosolic microdomains, would be totally blocked at ouabain concentrations necessary for limited binding to almost ouabain-insensitive a 1 -isoforms in the rat. References 1. Schatzmann, H J. (1953) Herzglycoside als Hemmstoffe fu ¨ rden aktiven Kalium- und Natriumtransport durch die Erythrocyten- membran. Helv. Physiol. Acta 11, 346–354. 2. Skou, J.C. (1960) Further investigations on a Mg 2+ +Na + -acti- vated adenosinetriphosphatase, possibly related to the active, linkedtransportofNa + and K + across the nerve membrane. Biochim. Biophys. Acta 42, 6–23. 3. Skou, J.C. (1965) Enzymatic basis for active transport of Na + and K + across cell membrane. Physiol. Rev. 45, 596–617. 4. Goto,A.,Yamada,K.,Yagi,N.,Yoshioka,M.&Sugimoto,T. (1992) Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol. Rev. 44, 377–399. 5. Hamlyn, J.M., Blaustein, M.P., Bova, S., DuCharme, D.W., Harris, D.W., Mandel, F., Mathews, W.R. & Ludens, J.H. (1991) Identification and characterization of a ouabain-like compound from human plasma. Proc. Natl. Acad. Sci. USA 88, 6259–6263. 6. Hansen, O. (1989) Characterization of fatty acid interaction with ouabain and vanadate binding to (Na + +K + ) -activated ATPase. Biochim. Biophys. Acta 986, 130–134. 7. Hansen, O. (1994) Do putative endogenous digitalis-like factors have a physiological role? Hypertension 24, 640. 8. Aizman, O., Uhle ´ n, P., Lal, M., Brismar, H. & Aperia, A. (2001) Ouabain, a steroid hormone that signals with slow calcium oscil- lations. Proc. Natl. Acad. Sci. USA 98, 13420–13424. 9. Haas, M., Askari, A. & Xie, Z. (2000) Involvement of Src and epidermal growth factor receptor in the signal-transducing func- tion of Na + /K + -ATPase. J. Biol. Chem. 275, 27832–27837. 10. Liu, J., Tian, J., Haas, M., Shapiro, J.I., Askari, A. & Xie, Z. (2000) Ouabain interaction with cardiac Na + /K + -ATPase initiates signal cascades independent of changes in intracellular Na + and Ca 2+ concentrations. J. Biol. Chem. 275, 27838– 27844. 11. Tian, J., Gong, X. & Xie, Z. (2001) Signal-transducing function of Na + -K + -ATPase is essential for ouabain’s effect on [Ca 2+ ] i in rat cardiac myocytes. Am. J. Physiol. 281, H1899–H1907. 12. Hansen, O. (1984) Interaction of cardiac glycosides with (Na + +K + ) -activated ATPase. A biochemical link to digitalis- induced inotropy. Pharmacol. Rev. 36, 143–163. 13. Scheiner-Bobis, G. & Schoner, W. (2001) A fresh facet for ouabain action. Nature Med. 7, 1288–1289. 14. Xie, Z. & Askari, A. (2002) Na,K-ATPase as a signal transducer. Eur. J. Biochem. 269, 2434–2439. 15. Schoner, W. (1993) Endogenous digitalis-like factors. In Progress in Drug Research 41 (Jucker, E., ed.), pp. 249–291. Birkha ¨ user Verlag, Basel. 16. Price, E.M. & Lingrel, J.B. (1988) Structure-function relationships in the Na,K-ATPase a subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry 27, 8400– 8408. 17. Crambert, G., Hasler, U., Beggah, A.T., Yu, C., Modyanov, N.N., Horisberger, J D., Lelie ` vre, L. & Geering, K. (2000) Transport and pharmacological properties of nine different human Na,K- ATPase isozymes. J. Biol. Chem. 275, 1976–1986. 18. Hansen, O. (2001) The a 1 isoform of Na + ,K + -ATPase in rat soleus and extensor digitorum longus. Acta Physiol. Scand. 173, 335–341. 19. Lu ¨ cking, K., Nielsen, J.M., Pedersen, P.A. & Jørgensen, P.L. (1996) Na-K-ATPase isoform (a 3 , a 2 , a 1 ) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding. Am. J. Physiol. 271, F253–F260. 20. Schoner, W. (2002) Endogenous cardiac glycosides, a new class of steroid hormones. Eur. J. Biochem. 269, 2440–2448. 21. Ferrandi, M., Manunta, P., Balzan, S., Hamlyn, J.M., Bianchi, G. & Ferrari, P. (1997) Ouabain-like factor quantification in mam- malian tissues and plasma. Comparison of two independent assays. Hypertension 30, 886–896. 22. Doris, P.A., Jenkins, L.A. & Stocco, D.M. (1994) Is ouabain an authentic endogenous mammalian substance derived from the adrenal? Hypertension 23, 632–638. 23. Orlowski, J. & Lingrel, J.B. (1988) Tissue-specific and develop- mental regulation of rat Na,K-ATPase catalytic a isoform and a subunit mRNAs. J. Biol. Chem. 263, 10436–10442. 24. McDonough, A.A., Zhang, Y., Shin, V. & Frank, J.S. (1996) Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes. Am.J.Physiol.270, C1221–C1227. 25.Lucchesi,P.A.&Sweadner,K.J.(1991)Postnatalchangesin Na,K-ATPase isoform expression in rat cardiac ventricle. J. Biol. Chem. 266, 9327–9331. 26. Hansen, O. (2000) Real concentrations of isoforms of Na,K- ATPase from western blots of tissue homogenates. In Na,K- ATPase and Related ATPases (Taniguchi,K.&Kaya,S.eds),pp. 131–138. Elsevier, Amsterdam. 27. Juhaszova, M. & Blaustein, M.P. (1997) Na + pump low and high affinity a subunit isoforms are differently distributed in cells. Proc. Natl. Acad. Sci. USA 94, 1800–1805. 28. Jewell, E.A. & Lingrel, J.B. (1991) Comparison of the substrate dependence properties of the rat Na,K-ATPase a1, a2, and a3 isoforms expressed in HeLa cells. J. Biol. Chem. 266, 16925–16930. Ó FEBS 2003 Endogenous ouabain in normal signal-transduction? (Eur. J. Biochem. 270) 1919 . a similar role by putative endogenous ouabain or OLF. Ouabain- sensitive and -insensitive a- isoforms of Na + /K + -ATPase in rats All isoforms of the catalytic. concentrations of ouabain. Plasma concentrations of putative endogenous ouabain In another recent minireview in Eur. J. Biochem.[20]. endogenous ouabain was claimed