Low-dose cardiotonic steroids increase sodium–potassium ATPase activity that protects hippocampal slice cultures from experimental ischemia

Neuroscience Letters 473 (2010) 67–71

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Low-dose cardiotonic steroids increase sodium–potassium ATPase activity that protects hippocampal slice cultures from experimental ischemia Martin Oselkin, Dezhi Tian, Peter J. Bergold ∗ Department of Physiology and Pharmacology, State University of New York-Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA

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Article history: Received 15 July 2009 Received in revised form 20 September 2009 Accepted 6 October 2009 Keywords: Cardiac glycosides Dose–response Neuroprotection Sodium pump isoforms

a b s t r a c t The sodium–potassium ATPase (Na/K ATPase) is a major ionic transporter in the brain and is responsible for the maintenance of the Na+ and K+ gradients across the cell membrane. Cardiotonic steroids such as ouabain, digoxin and marinobufagenin are well-characterized inhibitors of the Na/K ATPase. Recently, cardiotonic steroids have been shown to have additional effects at concentrations below their IC50 for pumping. The cardiotonic steroids ouabain, digoxin, and marinobufagenin all show an inverted U-shaped dose–response curve with inhibition of pumping at concentrations near their IC50 , while increasing Na/K ATPase activity at doses below their IC50 . This stimulatory effect of cardiotonic steroids was observed in vitro in hippocampal slice cultures as well as in the hippocampus in vivo. Increased Na/K ATPase activity has been shown to protect slice culture neurons from hypoxia–hypoglycemia. Ouabain protected slice culture neurons from experimental ischemia at concentrations that increased Na/K ATPase. This protective effect was observed when ouabain was dosed 30 min before, or 2 h following experimental ischemia. Ouabain no longer protected against experimental ischemia if the increase of Na/K ATPase was blocked. These data suggest that the protective effect of ouabain was due to increased Na/K ATPase activity. The demonstration of a neuroprotective effect of cardiotonic steroids could potentially assist in the treatment of stroke since digoxin, one of the cardiotonic steroids examined in this study, has approval by the Food and Drug Administration and can be safely administered at the concentrations that increase Na/K ATPase activity. © 2009 Elsevier Ireland Ltd. All rights reserved.

The Na/K ATPase is a major transporter in the brain that maintains the ionic gradients of Na+ and K+ [28]. Disruption of the sodium and potassium gradients depolarizes neurons and disables the Na/H, the Na/Ca and the Na/K/Cl transporters and sodium-dependent glutamate uptake [7]. All of these factors contribute to brain injury following ischemia [7]. Ischemic preconditioning protects the brain from ischemic injury. In preconditioning, a brief period of ischemia protects from a longer, damaging ischemic episode [29]. Cardiac ischemic preconditioning prevented the loss of Na/K ATPase activity that protected hearts from subsequent ischemia [8,18,20,22,27]. Preconditioning also prevented loss of Na/K ATPase in the kidney [1]. In the brain, ischemic preconditioning increased the activity of the Na/K ATPase [6]. In hippocampal slice cultures, increased pump activity protected neurons from an experimental ischemia of hypoxia–hypoglycemia [25]. Even though these studies implicate increased Na/K ATPase activity in protection against ischemia,

∗ Corresponding author at: Department of Physiology and Pharmacology, Box 29, State University of New York-Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, USA. Tel.: +1 718 270 3927; fax: +1 718 270 2241. E-mail address: [email protected] (P.J. Bergold). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.10.021

ischemic preconditioning is unlikely to be used in the clinic [24,29]. This study explores whether cardiotonic steroids are an alternative to ischemic preconditioning to increase basal Na/K ATPase and to test whether the increase in basal pumping protects neurons from ischemia. Low-dose cardiotonic steroids provide a potential way to increase Na/K ATPase activity. When dosed at concentrations near their IC50 , cardiotonic steroids inhibit the Na/K ATPase and are used in the clinic to treat congestive heart disease and arrhythmias [2,10]. There are scattered reports over many decades suggesting that cardiotonic steroids increased pumping at concentrations below their IC50 [3,5,11,12,14]. In addition, cardiotonic steroids are synthesized endogenously and are active at nanomolar concentrations [2,28]. This suggests that cardiotonic steroids are potentially regulators of Na/K ATPase. Recent studies have shown that, at high, pharmacological doses, cardiotonic steroids inhibit Na/K ATPase activity. At the lower, physiological concentrations that occur in vivo, cardiotonic steroids bind the Na/K ATPase and initiate cascades of intracellular signaling that could increase Na/K ATPase activity [2]. The Na/K ATPase is a heterodimer of ␣ and ␤ subunits [28]. A Na/K ATPase isoform is determined by which ␣ subunit it contains. Rat brain expresses three Na/K ATPase isoforms, ␣1 , ␣2 and

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Fig. 1. Ouabain, digoxin and marinobufagenin show an inverted U-shaped dose–response curve for Na/K ATPase. Na/K ATPase activity was assayed by 86 Rb uptake after drug or control treatment and normalized to control. Panel A: ouabain. Ouabain (120 nM) significantly increased pumping while ouabain (1200 nM) significantly decreased pumping (ANOVA, F3,23 = 38.37, p < 0.0001; post hoc test, ***p < 0.0001, **p < 0.01). Panel B: digoxin. Digoxin (10 nM) significantly increased pumping while digoxin (100 nM) significantly decreased pumping (ANOVA, F3,49 = 12.06, p < 0.0001; post hoc test, **p < 0.01, *p < 0.05). Panel C: marinobufagenin. MBG (100 pM) significantly increased pumping while MBG (1 nM) significantly decreased pumping (ANOVA, F3,33 = 12.24, p < 0.0001; post hoc test, p < 0.01, *p < 0.05). All values are average ± SEM.

␣3 [16,17]. Na/K ATPase isoforms bind cardiotonic steroids with differing affinities [2,9,21]. Digoxin, ouabain and marinobufagenin (MBG) are the cardiotonic steroids examined in this study. Digoxin and ouabain are members of the cardenolide family of cardiotonic steroids [2]. The ␣2 and ␣3 isoforms of Na/K ATPase bind ouabain and digoxin with higher affinity than the ␣1 isoform [9]. MBG is a member of the bufadienolide family [2]. MBG binds the ␣1 isoform of the Na/K ATPase with much higher affinity than the ␣2 and ␣3 [9]. This study examined whether ouabain, digoxin, or MBG increase Na/K ATPase activity at concentrations below their IC50 for pumping. In addition, hippocampal slice cultures were used to test whether cardiotonic steroids can protect against in vitro experimental ischemia. Hippocampal slice cultures were prepared as described by Hassen et al. [15]. Cultures were maintained for 2 weeks before experiments at 32 ◦ C in a 5% CO2 incubator. 86 Rb uptake (PerkinElmer, Waltham, MA) was done as described by Tian et al. [25]. Briefly, 86 RbCl (1 ␮Ci, >1 ␮Ci/␮g, PerkinElmer, Boston, MA) was added to the slice cultures in Earles Balanced Salt Solution con-

taining 2.3 mM KCl (2.3 mM K EBSS) for 30 min at 37 ◦ C. The reaction was stopped with 3 washes of ice-chilled 2.3 mM K EBSS and slices homogenized with 1 ml 0.1N NaOH. An aliquot of the homogenized sample was counted on a Beckman LS6000IC scintillation counter (Beckman Instruments, Fullerton, CA). Protein content was measured using a BCA assay (Pierce Chemical, Rockford, IL). Experimental ischemia was induced as described by Hassen et al. [15]. One day before an experiment, the cultures were shifted to a 37 ◦ C incubator in a 5% CO2 atmosphere. Experimental ischemia was induced by submerging slice cultures for 10 min in Earles Balanced Salt solution (BSS) without glucose that was bubbled vigorously with 95% N2 , 5% CO2 . A mock ischemia group was submerged in BSS with 5 mM glucose for 10 min and bubbled vigorously with 20% O2 , 75% N2 , 5% CO2 . Cell loss assays using propidium iodide (PI) were performed as described by Hassen et al. [15]. PI is a standard method to assay cell loss in slice cultures [19]. Briefly, slice cultures were incubated for 30 min with 0.5% PI and PI epifluorescence images were obtained with a CCD camera on a Zeiss Axiovert 100 microscope using rhodamine optics. Fluorescence images were analyzed using NIH Image J. Experiments were analyzed by one-way ANOVA.

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Differences were further analyzed by Bonferroni’s post hoc test. Statistical significance was set at 0.05. Unless indicated, all chemicals were purchased from Sigma, St. Louis, MO. MBG was kindly provided by Dr. Peter Doris (University of Texas at Houston, Houston, TX). Hippocampal 86 Rb uptake was performed ex vivo by interperitoneal (IP) injection of differing amounts of digoxin or vehicle into Sprague–Dawley rats (200–250 g, Charles River Laboratories, Wilmington, MA). After 30 min, the rats were lightly anesthetized with halothane, decapitated, the hippocampus isolated, and 400 ␮m transverse slices prepared. The slices were plated onto Millicell-CM filter inserts (Millipore, Billerica, MA) in 1 ml of BSS at 37 ◦ C. After 30 min, 86 Rb uptake was performed as described by Tian et al. [25]. To test whether ouabain, digoxin, or MBG increase in vitro Na/K ATPase activity differing concentrations of these compounds were applied to slice cultures followed by 86 Rb uptake assay (Fig. 1). Drugs were added to slice cultures for 60 min while control cultures were mock-treated. Thirty minutes after the drug addition, 86 Rb uptake was assayed. Doses of cardiotonic steroids below their IC50 were anticipated to have either no effect or increase pumping. The IC50 of ouabain for rodent brain ␣1 , ␣2 and ␣3 Na/K ATPase isoforms are: 1.3 mM, 4.5 ␮M and 2.9 ␮M, respectively [17]. Therefore, slice cultures were treated with 15 nM, 120 nM or 1.2 ␮M ouabain (Fig. 1A). Ouabain (1.2 ␮M), a dose close to its IC50 for the ␣2 and ␣3 isoform of Na/K ATPase partially inhibited pumping. In contrast, ouabain (120 nM) significantly increased 86 Rb uptake. Ouabain (15 nM) had no effect suggesting that the increase in 86 Rb uptake was dose-dependent. To confirm that ouabain (120 nM) increased Na/K ATPase activity, slice cultures were treated for 60 min with ouabain (120 nM) to increase pumping or mocktreated. Ouabain (120 nM) was washed out and 86 Rb uptake was performed in the presence of ouabain (2 mM), a concentration that inhibits all rat brain Na/K ATPase isoforms [17]. Greater than 90.2 ± 2.0% (n = 4) of the 86 Rb uptake induced by ouabain (120 nM) was blocked by ouabain (2 mM) suggesting that the increase in 86 Rb uptake was due to increased Na/K ATPase activity. Digoxin inhibits the rat brain Na/K ATPase with an IC50 of 130 ␮M, 25 nM and 25 nM for the ␣1 , ␣2 and ␣3 , respectively [4]. Digoxin was tested at 1 nM, 10 nM and 100 nM (Fig. 1B). Digoxin (100 nM) partially inhibited 86 Rb uptake. In contrast, digoxin (10 nM) increased 86 Rb uptake. The increase in 86 Rb uptake was dose-dependent since digoxin (1 nM) had no effect. The increased 86 Rb uptake by digoxin (10 nM) was inhibited 92.0 ± 0.4% (n = 7) by ouabain (2 mM) suggesting that the increased 86 Rb uptake was due to basal Na/K ATPase activity. MBG is a member of the bufadienolide family that is chemically distinct from the ouabain and digoxin [2]. The IC50 of MBG for rat Na/K ATPase activity is 2.6 nM, 50 nM and 140 nM for the ␣1 , ␣2 and ␣3 , isoforms [9]. MBG was dosed at 10 pM, 100 pM and 1 nM to slice cultures. MBG (1 nM) inhibited 86 Rb uptake while MBG (100 pM) significantly increased 86 Rb uptake. This increase was dose-dependent since MBG (10 pM) had no effect. Ouabain (2 mM) blocked 91.6 ± 6.1% (n = 6) of the increased 86 Rb uptake induced by MBG (100 pM). These data suggests that both cardenolides and bufadienolides increase basal Na/K ATPase activity at concentrations below their IC50 for pumping. Low-dose digoxin increased Na/K ATPase activity in vitro (Fig. 1). To examine if digoxin acts similarly in vivo, rats received IP injections of 650, 130, or 65 ␮g/kg digoxin. Control rats received an equivalent vehicle injection. The digoxin doses were calculated from a previous study that analyzed brain digoxin levels following IP injection into Sprague–Dawley rats [13]. Intraperitoneal injections of 650, 130 or 65 ␮g/kg corresponded to a dose above, equivalent, and below its IC50 for Na/K ATPase. Na/K ATPase activity was assayed by 86 Rb uptake 60 min after injection (Fig. 2). At

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Fig. 2. Digoxin increases Na/K ATPase in vivo. Rats were injected IP with differing doses of digoxin. After 30 min, the hippocampus was removed, sliced into 400 ␮m transverse slices and Na/K ATPase activity assayed by 86 Rb uptake. Digoxin (65 ␮g/kg) significantly increased Na/K ATPase activity over mock-treated (ANOVA, F3,38 = 4.23, p < 0.001, post hoc test,**p < 0.001) Values are mean ± SEM.

65 ␮g/kg, digoxin increased Na/K ATPase, 130 ␮g/kg had no effect, and 650 ␮g/kg inhibited Na/K ATPase activity. The increased 86 Rb uptake was inhibited 88.5 ± 5.2% (n = 5) by ouabain (2 mM) suggesting that the digoxin (65 ␮g/kg) injection increased Na/K ATPase activity. The dose–response of digoxin in vivo was similar to the inverted U-shaped dose–response observed using slice cultures. Ouabain (120 nM) treatment increased pumping in slice cultures (Fig. 1). Ischemic preconditioning also increased in Na/K ATPase activity and this increase in pumping protected slice cultures against experimental ischemia [25]. We tested whether ouabain (120 nM) also protected slice cultures from experimental ischemia. Slice cultures were treated with ouabain (120 nM) or vehicle for 30 min, the drugs were washed out, and the cultures received mock or experimental ischemia. After mock or experimental ischemia, the cultures were returned to the incubator for 3 days followed by PI assay. Mock ischemia induced minimal PI fluorescence in cultures receiving vehicle or ouabain (120 nM) (Fig. 3). Experimental ischemia increased PI fluorescence and the PI fluorescence increase induced by experimental ischemia was significantly reduced by ouabain (120 nM) (Fig. 3A and B). To further test whether increased Na/K ATPase activity was needed to protect slice cultures, MBG (2 nM) and dihydroouabain (DHO) were used as specific inhibitors of the ␣1 or ␣2 /␣3 Na/K ATPase isoforms, respectively [9,23]. MBG (2 nM) and DHO (20 ␮M) also blocked both the increase in Na/K ATPase and the neuroprotective effect induced by preconditioning in slice cultures [25]. Slice cultures received ouabain (120 nM) treatment for 30 min; after washout, the cultures received experimental ischemia in the presence of MBG (2 nM) or DHO (20 ␮M). The MBG or DHO were washed out 20 min after mock or experimental ischemia and PI fluorescence was assayed 3 days later. MBG (2 nM) treatment significantly blocked the reduction of PI fluorescence by ouabain (120 nM) suggesting that MBG (2 nM) blocked a protective effect of ouabain (120 nM) (Fig. 3B). In contrast, DHO (20 ␮M) had no significant effect on the ouabain (120 nM) reduction of PI fluorescence. These data suggest that slice cultures were protected by the increase in basal Na/K ATPase activity induced by ouabain (120 nM). These data also suggest that the ␣1 isoform of Na/K ATPase underlies the protective effect of ouabain

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Fig. 3. Low-dose ouabain protects slice cultures when dosed before experimental ischemia. Slice cultures were treated with either ouabain (120 nM) or vehicle for 30 min. After washout, the cultures received 10-min of mock or experimental ischemia. The cultures were maintained in the incubator for 3 days and PI fluorescence in the pyramidal cell layer was assayed by NIH Image J software. A subset of cultures treated with ouabain (120 nM) were also treated with MBG (2 nM) or DHO (20 ␮M) during experimental ischemia. Panel A: representative fluorescent images of slice cultures that were treated with vehicle or ouabain (120 nM) before experimental ischemia. PI fluorescence was limited to the pyramidal and granule layers of the slice cultures (dotted line). Panel B: summary of these experiments. PI fluorescence in the ouabain (120 nM) treated cultures was significantly less than in vehicle-treated cultures (ANOVA, F5,34 = 20.7, p < 0.001, post hoc, **p < 0.001) n = 6 for all groups except n = 5 for the group receiving vehicle treatment and experimental ischemia. PI fluorescence in the group receiving ouabain (120 nM) followed by MBG (2 nM) was significantly increased as compared to ouabain (120 nM) alone (post hoc, *p < 0.05). Values are mean ± SEM.

(120 nM) since it could be blocked with MBG (2 nM) but not DHO (20 ␮M). Ouabain (120 nM) limited PI fluorescence when applied before experimental ischemia (Fig. 3), therefore it was tested whether it was effective when administered 2 h after ischemia (Fig. 4). Slice cultures were treated with ouabain (120 nM) or vehicle 2 h after mock or experimental ischemia. PI fluorescence was assayed 3 days later. PI fluorescence was significantly lower in the ouabain (120 nM) group than the vehicle-treated group (Fig. 4A and B). These data suggest that ouabain protects neurons when applied 2 h following experimental ischemia. This study examined whether cardiotonic steroids increased Na/K ATPase in hippocampal slice cultures and whether this increased Na/K ATPase protected against experimental ischemia. Ouabain, digoxin and marinobufagenin had an inverted U-shaped dose–response to Na/K ATPase (Fig. 1). All three cardiotonic steroids inhibited basal pump activity when dosed at concentrations close to their IC50 for Na/K ATPase. Unexpectedly, at doses 10–100-fold below their IC50 , all three compounds increased basal Na/K ATPase activity (Fig. 1). This stimulatory effect was not limited to slice cultures in vitro, digoxin also increased the basal activity of Na/K ATPase in vivo (Fig. 2). Ouabain (120 nM) protected slice culture neurons when dosed either before or after experimental ischemia (Figs. 3 and 4). MBG (2 nM) blocked the protective effect of ouabain (120 nM) suggesting that an increased activity of the ␣1 isoform of Na/K ATPase was responsible for the protective effect of ouabain (120 nM) (Fig. 3). Ouabain, digoxin and MBG increased Na/K ATPase activity in slice cultures (Fig. 1). The molecular mechanism of how low-doses of these cardiotonic steroids increase Na/K ATPase is unknown, however a likely explanation is that it involved the induction of intracellular signaling from the Na/K ATPase [2]. At concentrations below its IC50 for pumping, binding of ouabain to the renal Na/K ATPase activated a multiprotein complex on the Na/K ATPase that

Fig. 4. Low-dose ouabain protects slice cultures when dosed after experimental ischemia Slice cultures received 10 min of mock or experimental ischemia and returned to the incubator for 2 h, followed by either vehicle or ouabain (120 nM) treatment for 30 min. PI fluorescence was assayed three days later. Panel A: representative fluorescent images of slice cultures that were treated with vehicle or ouabain (120 nM) after experimental ischemia. PI fluorescence was limited to the pyramidal and granule layers of the slice cultures (dotted line). Panel B: summary of these experiments. PI fluorescence in the cultures receiving ouabain (120 nM) was significantly less than in vehicle-treated cultures (ANOVA, F4,23 = 25.3, p < 0.0001; n = 6 for all groups, post hoc, **p < 0.0001). Values are mean ± SEM.

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includes src, ras, mitogen-activated protein kinases, and the epidermal growth factor receptors [2,28]. This activation of intracellular signaling has been most extensively studied in renal epithelia, however, many of the same signaling proteins are expressed in brain [28]. In brain, cardiotonic steroids may increase Na/K ATPase through a similar set of intracellular signals. Ischemic preconditioning increased Na/K ATPase activity in brain, heart and kidney [1,6,8,18,20,22,27]. This increased Na/K ATPase activity also protected against experimental ischemia in slice cultures (Fig. 3B) [25]. Ischemia disrupts ionic gradients across the cell membrane [7]. Increased Na/K ATPase activity could either prevent the loss of ionic gradients or promote a more rapid restoration of pumping after blood flow resumes. Low-dose cardiotonic steroids, including digoxin, protected neurons in cortical brain slices from experimental ischemia, however, the mechanism of the protective effect of cardiotonic steroids was not examined [26]. The results of this study suggest that the neuroprotective effect of lowdose cardiotonic steroids may result from increased Na/K ATPase activity. References [1] C. Aufricht, B. Bidmon, D. Ruffingshofer, H. Regele, K. Herkner, N.J. Siegel, M. Kashgarian, S.K. Van Why, Ischemic conditioning prevents Na,K-ATPase dissociation from the cytoskeletal cellular fraction after repeat renal ischemia in rats, Pediatr. Res. 51 (2002) 722–727. [2] A.Y. Bagrov, J.I. Shapiro, O.V. Fedorova, Endogenous cardiotonic steroids: physiology, pharmacology, and novel therapeutic targets, Pharmacol. Rev. 61 (2009) 9–38. [3] P.K Boyer, C.A. Poindexter, The influence of digitalis on the electrolyte and water balance of heart muscle, Am. Heart J. 20 (1940) 556–591. [4] N.J. Cano, A.E. Sabouraud, M. Debray, J.-M.G. Scherrmann, Dose-dependent reversal of digoxin-inhibited activity of an in vitro Na+ K+ ATPase model by digoxin-specific antibody, Toxicol. Lett. 85 (1996) 107–111. [5] I. Cohen, J. Daut, D. Noble, An analysis of the actions of low concentrations of ouabain on membrane currents in Purkinje fibers, J. Physiol. 260 (1976) 75–103. [6] A.T. de Souza Wyse, E.L. Streck, P. Worm, A. Wajner, F. Ritter, C.A. Netto, Preconditioning prevents the inhibition of Na+ ,K+ -ATPase activity after brain ischemia, Neurochem. Res. 25 (2000) 971–975. [7] K.P. Doyle, R.P. Simon, M.P. Stenzel-Poore, Mechanisms of ischemic brain damage, Neuropharmacology 55 (2008) 310–318. [8] A.B. Elmoselhi, A. Lukas, O. Ostadal, N.S. Dhalla, Preconditioning attenuates ischemia–reperfusion-induced remodeling of Na+ -K+-ATPase in hearts, Am. J. Physiol. Heart Circ. Physiol. 285 (2003) H1055–H1063. [9] O.V. Fedorova, A.Y. Bagrov, Inhibition of Na/K ATPase from rat aorta by two Na/K pump inhibitors, ouabain and marinobufagenin: evidence of interaction with different alpha-subunit isoforms, Am. J. Hypertens. 10 (1997) 929–935. [10] M Gheorghiade, K.F. Adams Jr., W.S. Colucci, Digoxin in the management of cardiovascular disorders, Circulation 109 (2004) 2959–2964.

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