Georgios Scheiner-Bobis

Endogenous and Exogenous Cardiac Glycosides and their Mechanisms of Action

Cardiac glycosides have been used for decades to treat congestive heart failure. The recent identification of cardiotonic steroids such as ouabain, digoxin, marinobufagenin, and telocinobufagin in blood plasma, adrenal glands, and hypothalamus of mammals led to exciting new perspectives in the pathology of heart failure and arterial hypertension. Biosynthesis of ouabain and digoxin occurs in adrenal glands and is under the control of angiotensin II, endothelin, and epinephrine released from cells of the midbrain upon stimulation of brain areas sensing cerebrospinal Na+ concentration and, apparently, the body’;s K+ content. Rapid changes of endogenous ouabain upon physical exercise may favor the economy of the heart by a rise of intracellular Ca2+ levels in cardiac and atrial muscle cells. According to the sodium pump lag hypothesis, this may be accomplished by partial inhibition of the sodium pump and Ca2+ influx via the Na+/Ca2+ exchanger working in reverse mode or via activation of the Na+/K+-ATPase signalosome complex, generating intracellular calcium oscillations, reactive oxygen species, and gene activation via nuclear factor-κB or extracellular signal-regulated kinases 1 and 2. Elevated concentrations of endogenous ouabain and marinobufagenin in the subnanomolar concentration range were found to stimulate proliferation and differentiation of cardiac and smooth muscle cells. They may have a primary role in the development of cardiac dysfunction and failure because (i) offspring of hypertensive patients evidently inherit elevated plasma concentrations of endogenous ouabain; (ii) such elevated concentrations correlate positively with cardiac dysfunction, hypertrophy, and arterial hypertension; (iii) about 40% of Europeans with uncomplicated essential hypertension show increased concentrations of endogenous ouabain associated with reduced heart rate and cardiac hypertrophy; (iv) in patients with advanced arterial hypertension, circulating levels of endogenous ouabain correlate with BP and total peripheral resistance; (v) among patients with idiopathic dilated cardiomyopathy, high circulating levels of endogenous ouabain and marinobufagenin identify those individuals who are predisposed to progressing more rapidly to heart failure, suggesting that endogenous ouabain (and marinobufagenin) may contribute to toxicity upon digoxin therapy.In contrast to endogenous ouabain, endogenous marinobufagenin may act as a natriuretic substance as well. It shows a higher affinity for the ouabain-insensitive α1 isoform of Na+/K+-ATPase of rat kidney tubular cells and its levels are increased in volume expansion and pre-eclampsia. Digoxin, which is synthesized in adrenal glands, seems to counteract the hypertensinogenic action of ouabain in rats, as do antibodies against ouabain, for example, (Digibind®) and rostafuroxin (PST 2238), a selective ouabain antagonist. It lowers BP in ouabain- and adducin-dependent hypertension in rats and is a promising new class of antihypertensive medication in humans.

A fresh facet for ouabain action

Ouabain signaling through a plasma membrane can produce oscillations of intracellular calcium levels, resulting in translocation of the NF-κB transcription factor into the nucleus and gene activation. This is a previously unrecognized form of steroid action.

Role of endogenous cardiotonic steroids in sodium homeostasis

Consumption of an excess of salt over many years leads to arterial hypertension [1]. This process is accompanied by the activation of a great number of genes involved in the remodelling and hypertrophy of the heart, kidneys and the wall of the arteries. These sodium-induced alterations lead to a higher incidence of stroke, greater stiffness of conduit arteries and enhanced activity of resistance arteries [1–3]. Increased uptake of sodium from the diet may not, in some persons, affect the levels of circulating renin, angiotensin and norepinephrine in blood plasma and increase the urinary secretion of sodium, potassium and calcium [4]. Sodium sensors [5] and natriuretic hormones must be involved in the control of the body’s sodium content [6,7]. Defects in this control mechanism induced by an altered gene expression pattern in different organs may explain why excess sodium consumption is toxic [2]. The recently discovered new class of endogenous cardiotonic steroid hormones involved in the control of heart function, vasoconstriction and kidney function seems to shed new light on the sodium toxic mechanisms that are more prominent in situations such as kidney failure and uraemic cardiomyopathy. The identification of new mechanisms inducing arterial hypertension may also open up the possibility of new therapeutic approaches [8,9]. The search for an additional natriuretic hormone involved in low-renin hypertension was initiated [6,10,11] because the existence of such an hormone was evident and not all of the Na+-related effects on the control of fluid volume and osmolality by the kidney, the circulatory system and the heart could be explained [7,12,13]. Hence, the idea evolved that a circulating inhibitor of the sodium pump of renal tubular cells might exist. Such a substance should act like an endogenous digitalis [13]. In fact, a close correlation between blood pressure and the concentration of a circulating inhibitor of the sodium pump in human blood plasma was

Involvement of the M7/M8 Extracellular Loop of the Sodium Pump α Subunit in Ion Transport STRUCTURAL AND FUNCTIONAL HOMOLOGY TO P-LOOPS OF ION CHANNELS

Mutations were introduced in the motif 884DDRW887 from an extracellular peptide of the sodium pump α subunit localized between M7 and M8 membrane spans to investigate a possible role of this structure in ion recognition. A homologous sequence 399QDCW402that occurs in the P-loops of Na+ channels was shown earlier to be important for ion gating. Mutant sodium pumps were expressed in yeast and subsequently investigated for their behavior toward ouabain, Na+, K+, and ATP. Native enzyme and D884A, D884R, D885A, D885E, or D885R mutants all bind ouabain in the presence of phosphate and Mg2+. TheK D values determined from Scatchard analysis are in the range 5–8 nm for the native enzyme and the D884A, D885E, or D885A mutants, and 15.7 ± 2.04 and 30.1 ± 4.32 nm for mutants D884R and D885R, respectively. This ouabain binding is reduced in the presence of K+ in a similar way for both native or mutant sodium pumps with relative affinities (K 0.5) for K+ ranging from 1.4 to 3.7 mm. Ouabain binding in the presence of 100 μm ATP is promoted by Na+ withK 0.5 = 1.64 ± 0.01 mm for the native enzyme and K 0.5 = 8.6 ± 1.35 mm for the D884R mutant. The K 0.5values of the two enzymes for ATP are 0.66 ± 0.16 μm and 1.1 ± 0.12 μm, respectively. Ouabain binding as a function of Na+ concentration, on the other hand, is very low for the D885R mutant, even at an ATP concentration of 2 mm. Phosphate or eosin, however, are recognized by this mutant enzyme, so that a major conformational change within the ATP-binding site appears unlikely. The inability of the D885R mutant to bind ouabain in the presence of Na+ and ATP could be explained by assuming that the M7/M8 connecting extracellular loop, which also contains the mutated amino acids, is invaginated within the plane of the plasma membrane and possibly involved in acceptance and/or release of Na+ ions coming from cytosolic areas of the protein. In this case, the placement of an additional positive charge might repel Na+ ions and interrupt their flow, thus not allowing the enzyme to assume the proper conformational state for ouabain binding. Such invaginated hydrophilic protein structures, such as the P-loops of Na+ and K+ channels, are already known and have been shown to participate in ion conduction.

Dehydroepiandrosterone sulfate mediates activation of transcription factors CREB and ATF-1 via a Gα11-coupled receptor in the spermatogenic cell line GC-2.

Dehydroepiandrosterone sulfate (DHEAS) is a circulating steroid produced in the adrenal cortex, brain, and gonads. Whereas a series of investigations attest to neuroprotective effects of the steroid in the brain, surprisingly little is known about the physiological effects of DHEAS on cells of the reproductive system. Here we demonstrate that DHEAS acting on the spermatogenic cell line GC-2 induces a time- and concentration-dependent phosphorylation of c-Src and Erk1/2 and activates the transcription factors activating transforming factor-1 (ATF-1) and cyclic AMP-responsive element binding protein (CREB). These actions are consistent with the non-classical signaling pathway of testosterone and suggest that DHEAS is a pro-androgen that is converted into testosterone in order to exert its biological activity. The fact, however, that steroid sulfatase mRNA was not detected in the GC-2 cells and the clear demonstration of DHEAS-induced activation of Erk1/2, ATF-1 and CREB after silencing the androgen receptor by small interfering RNA (siRNA) clearly contradict this assumption and make it appear unlikely that DHEAS has to be converted in the cytosol into a different steroid in order to activate the kinases and transcription factors mentioned. Instead, it is likely that the DHEAS-induced signaling is mediated through the interaction of the steroid with a membrane-bound G-protein-coupled receptor, since silencing of Guanine nucleotide-binding protein subunit alpha-11 (Gnα11) leads to the abolition of the DHEAS-induced stimulation of Erk1/2, ATF-1, and CREB. The investigation presented here shows a hormone-like activity of DHEAS on a spermatogenic cell line. Since DHEAS is produced in male and female reproductive organs, these findings could help to define new roles for DHEAS in the physiology of reproduction.

ION-TRANSPORTING ATPASES AS ION CHANNELS

Ion-transporting ATPases (pumps) hydrolyze ATP to maintain ion gradients across cell membranes. A presupposition for the maintenance of the gradients is that the ionophore of the pump that conducts the ions is accessible only from one of the two surfaces of the plasma membrane at any given time. Thus, a characteristic feature of pumps is an occluded state of the transported ions, whereas ion channels upon stimulation remain open at both ends and allow ions to flow through them down their chemical gradients. Recent experiments, however, provide evidence that a channel, simultaneously open on both sides of the plasma membrane, can also be formed within the mammalian sodium pump (Na+,K+-ATPase) upon its interaction with the marine toxin palytoxin, thus underlining common structural features shared by channels and pumps. This assumption is further supported by the demonstration of structural and functional homology between the extracellular loop of the sodium pump α subunit connecting the M7 and M8 transmembrane spans and the P-loops of Na+ channels. Possibly, pumps are simply channels that are able to be gated by ATP and its product phosphate.

Is the Sodium Pump a Functional Dimer

It is presently unclear whether the membrane-embedded sodium pump works as an (αs) monomer (4) according to the single site model (15,32) or as an (αs)2 diprotomer of interacting α subunits (21–23,33). The single site model includes as an essential step the conversion of the low affinity ATP binding site (E2ATP site) to the high affinity ATP binding site (E1ATP site). This model implies that high and low affinity ATP binding sites cannot exist simultaneously, since they are two different forms of the same ATP binding site that can exist either in the E1ATP or the E2ATP conformational state. There are a number of data which are difficult to reconcile with the idea that the membrane-embedded sodium pump works according to the single site model as an (αs) monomer: (a) a negative cooperativity in ATP hydrolysis is seen (3,24) which may indicate an interaction of ATP binding sites; this is also seen under conditions when the catalytically active (αs) protomer of the sodium pump is existent as shown by active-enzyme analytical centrifugation of detergent solubilized Na+/K+-ATPase (37). (b) radiation inactivation reveals a bigger target size for the overall reaction than for partial reactions of the pump (7, 16, 17); (c) the enzyme shows the phenomenon of superphosphorylation at high concentrations of ATP (20); (d) simultaneous binding of TNP-ADP and phosphate to the FITC-modified enzyme have been recorded (27) (FITC binds to the high affinity ATP binding site in the E1ATP state); (e) Forster energy transfer measurements between the FITC-site and an ATP-protectable erythrosin isothiocyanate-site reveals that both sites are 5.5 nm apart (2); (f) Consistent with the idea that the onset of turnover of the sodium pump might cause (αs)protomers to form (αs)2 diprotomers is the observation that various concentrations of ATP, protein and phosphatidylserine affect the (αs) protomer ⇌ (αs)2 diprotomer equilibrium (14,10).

Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump.

The α4 isoform of the Na(+),K(+)-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.

Non-classical testosterone signaling in spermatogenic GC-2 cells is mediated through ZIP9 interacting with Gnα11 ☆

Although classical and non-classical signaling of testosterone has been documented in several investigations, the nature of the receptor involved in the non-classical pathway remains a source of controversy. While some investigators favor the exclusive participation of the cytosolic/nuclear androgen receptor (AR) in both pathways, others propose a membrane-bound receptor as the mediator of the non-classical testosterone signaling. Evidence is provided here that in the spermatogenic cell line GC-2 the non-classical signaling pathway of testosterone, characterized through the activation of Erk1/2 and transcription factors like CREB or ATF-1, is not mediated through the classical nuclear androgen receptor (AR) but rather by a membrane-associated receptor. This receptor is ZIP9, a Zn(2+) transporter from the family of the ZRT, IRT-like proteins (ZRT=zinc-regulated transporter; IRT=iron-regulated transporter), which directly interacts with the G-protein Gnα11. siRNA-induced abrogation of the expression of either of these two proteins, whose close contacts are demonstrated by an in situ proximity assay, completely prevents all non-classical signaling effects of testosterone addressed. In contrast, silencing of AR expression does not influence the same signaling events. The identification of ZIP9/Gnα11 interactions as the mediators of the non-classical testosterone signaling cascade in spermatogenic GC-2 cells might help to supplement our knowledge concerning the role of testosterone in male fertility and reproduction.

Non-classical testosterone signaling mediated through ZIP9 stimulates claudin expression and tight junction formation in Sertoli cells

In the classical signaling pathway, testosterone regulates gene expression by activating the cytosolic/nuclear androgen receptor. In the non-classical pathway, testosterone activates cytosolic signaling cascades that are normally triggered by growth factors. The nature of the receptor involved in this signaling pathway is a source of controversy. In the Sertoli cell line 93RS2, which lacks the classical AR, we determined that testosterone stimulates the non-classical signaling pathway, characterized by the phosphorylation of Erk1/2 and transcription factors CREB and ATF-1. We also demonstrated that testosterone increases the expression of the tight junction (TJ) proteins claudin-1 and claudin-5. Both of these proteins are known to be essential constituents of TJs between Sertoli cells, and as a consequence of their increased expression transepithelial resistance across Sertoli cell monolayers is increased. ZIP9 is a Zn(2+)transporter that was recently shown to be a membrane-bound testosterone receptor. Silencing its expression in 93RS2 Sertoli cells by siRNA completely prevents Erk1/2, CREB, and ATF-1 phosphorylation as well the stimulation of claudin-1 and -5 expression and TJ formation between neighboring cells. The study presented here demonstrates for the first time that in Sertoli cells testosterone acts through the receptor ZIP9 to trigger the non-classical signaling cascade, resulting in increased claudin expression and TJ formation. Since TJ formation is a prerequisite for the maintenance of the blood-testis barrier, the testosterone/ZIP9 effects might be significant for male physiology. Further assessment of these interactions will help to supplement our knowledge concerning the mechanism by which testosterone plays a role in male fertility.