Endogenous Cardiac Glycosides

Endogenous Cardiac Glycosides Ralph A. Kelly and Thomas W. Smith Cardiovascular Division Department of Medicine Brigham and Women’s Hospital and Haruard Medical School Boston, Massachusetts 02115

1. Introduction The cardiac glycosides comprise a class of organic compounds derived largely from plant sources that have been used for millennia as therapeutic agents. In modem times the use of digitalis glycosides was described and codified in William Withering’s 1785 monograph on the therapeutic efficacy of the leaves of the common foxglove plant, Digitalis purpurea (Withering, 1941). Other clinically relevant cardiac glycosides are derived from the leaves of Digitalis lanata, from which digitoxin and digoxin are derived, and from the seeds of Strophanthus gratus, which contain Gstrophanthin (ouabain). Indeed, the molecular motif characteristic of the cardiac glycosides-a steroid nucleus containing an a,&unsaturated lactone ring at the C1, position-are found in a large number of plants and a few toad species, usually acting as a venom or poison serving to alter the future behavior of predators (Guntert and Linde, 1981). The bufodienolides, for example, are a well-characterized family of digitalis-like compounds present in the venom and skin of some toads that include the characteristic cyclopentanephenathrene steroid nucleus, but with a sixmembered doubly unsaturated lactone ring at C1,, either with or without (i-e., a genin or aglycone) a sugar moiety. The affinity of cardiac glycosides for their molecular target or “receptor,” the plasmalemmal NaK-ATPase, has traditionally been Advances in Pharmacology. Volume 25 Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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thought to require the presence of an unsaturated lactone ring at C,,, a p-hydroxyl group at C,4, and cis fusion of the A-B and C-D rings of the steroid nucleus. However, synthetic derivatives of endogenous steroid hormones have been shown to have “cardiotonic” biological activity despite the trans configurations of the ring junctions. Indeed, 14/3-hydroxyprogesteronehas approximately one-tenth the potency of the aglycone congener of ouabain and does exhibit positive inotropic properties in isolated cardiac muscle preparations (for a review see LaBelle et al., 1989). Thomas er al. (1989) and Repke e? a / . (1991) have recently argued for a reconsideration of the constraints imposed by the structure-activity relationships dictated by the classic cardiac glycosides as being unduly restrictive. The absence of the C,, unsaturated lactone, as well as the sugar moiety, for example, merely shift the concentrationeffect relationship for digitalis-like inhibition of NaK-ATPase activity. Despite the rapid pace of development of new drugs for the treatment of cardiovascular disease in the latter half of this century, the cardiac glycosides remain among the most common drugs prescribed by physicians throughout the world. While less commonly used now as first-line agents in the treatment of supraventricular dysrhythmias, due principally to the development of other effective and potentially less toxic therapeutic modalities, cardiac glycosides remain the only effective and relatively safe positively inotropic agents with acceptable oral bioavailability suitable for long-term administration. The reader is referred to several recent reviews that reaffirm the utility and safety of these drugs for the treatment of systolic ventricular dysfunction (Kelly and Smith, 1993a,b).

11. Digitalis-like Factors as Natriuretic Hormones Over the past two decades many reports have appeared providing evidence for the possibility that endogenous ligands exist for the cardiac glycoside binding site on NaK-ATPase, the “sodium pump” present within the plasmalemmal membranes of animals as phylogenetically diverse as brine shrimp and mammals. Ironically, the postulated physiological role(s) of a digitalis-like hormone or autacoid has nothing to do with the accepted pharmacological indications for the cardiac glycosides, that is, as an antidysrhythmic and cardiotonic agent. Research in the 1950s and 1960s supported the hypothesis that, in addition to aldosterone and antidiuretic hormone, there existed a “third factor” that regulated sodium and water homeostasis, a “natriuretic hormone.” As active sodium transport by sodium pumps in the basolateral membranes of renal tubular epithelial cells is responsible for as much as one-half of the sodium reabsorbed

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along the nephron (Katz, 1982; Doucet, 1992), this led to the suggestion that a circulating inhibitor of NaK-ATPase would be an effective natriuretic hormone. Although the discovery of the atrial natriuretic peptides and related congeners in the 1980s undoubtedly explained some portion of the physiological observations that predicted the existence of endogenous natriuretic factors, this did not exclude the possibility of other natriuretic mechanisms, as discussed by dewardener (1991), a renal physiologist who was among the first to provide experimental evidence for natriuretic hormones. Additional evidence for a role of such a factor in volume regulation came from observations by Smith and Welt (1970) that sodium pump function was depressed in patients with end-stage renal disease. As this physiological condition is often characterized by abnormal extracellular volume expansion due to the loss of functional nephrons, this should provide a marked stimulation for release of a natriuretic hormone into the circulation and inhibition of NaK-ATPase activity in a variety of tissues. While many reports have appeared in the literature over the past 30 years documenting abnormalities of cellular ion homeostasis in uremic patients and in experimental animal models of renal failure, it is still unclear to what extent this is due to the multiple metabolic abnormalities that are characteristic of this syndrome, and whether the presence in excess of a circulating specific inhibitor of NaK-ATPase plays any role. In addition to its potential role as a natriuretic hormone, a second rationale for the existence of an endogenous digitalis-like compound has centered on its potential role in the regulation of blood pressure. By reducing the turnover rate of NaK-ATPase present in the sarcolemmal membrane of vascular smooth muscle in resistance vessels, this would result in vasoconstriction, possibly by increasing intracellular Ca2+activity due to increased influx of Ca2+through voltage-sensitive channels, as suggested by Haddy and Overbeck (1976), or by decreased Ca2+ efflux via the sarcolemmal Na+/Ca2+antiporter, as first postulated by Blaustein (1993). These hypotheses provided an important theoretical link between sodium homeostasis and the pathophysiology of hypertension. If an endogenous inhibitor of NaK-ATPase acting as a natriuretic hormone were released into the circulation in response to an increase in extracellular volume, the resulting natriuresis would be accompanied by an increase in peripheral vascular resistance, an increase that would be sustained if the stimulus for release (e.g., a high dietary sodium intake) persisted. This potential association has led to the appearance of numerous reports providing evidence of an endogenous digitalis-like inhibitor of NaKATPase in patients with hypertension and in experimental animal models. Most of this evidence antedates the recent identificationof specific endogenous inhibitors of NaK-ATPase (discussed in more detail below). The

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reader is referred to a number of comprehensive reviews of the earlier literature (dewardener, 1991 ; dewardener and Clarkson, 1985; Goto et al., 1992; Kelly and Smith, 1989; Cloix and Kelly, 1993; Haddy, 1991; Hamlyn and Manunta, 1992; Haupert, 1988; Graves and Williams, 1987; Schoner, 1991; Blaustein and Hamlyn, 1991; Blaustein, 1993).

Ill. NaK-ATPase as a ”Receptor”: Is the Analogy Appropriate?

Another argument often cited as evidence of the existence of an endogenous digitalis-like hormone or autacoid is the persistence of the cardiac glycoside “binding site,” on the a-subunit of NaK-ATPase throughout most of the phylogeny of eukaryotes. This could be due simply to an absolute requirement for a particular amino acid sequence and conformation of the enzyme necessary for successful ion translocation that had nothing to do with binding a putative natriuretic hormone, but which has provided a target for evolutionary selection favoring certain plants and toads as a means of poisoning animal predators. Other explanations for the persistent expression of the cardiac glycoside binding site throughout evolution have been articulated, but remain purely speculative (Kelly and Smith, 1989). The affinity of the a-subunit for the cardiac glycosides, by convention usually described in terms of the binding affinity of ouabain, which is relatively water soluble and therefore convenient for in uitro experiments, varies among species and among the three known a-subunit isoforms, each of which is encoded by a separate gene. Several reports have now appeared that emphasize the importance of the amino acid composition of the extracellular domain between the H1 and H2 transmembrane domains of the a,-subunit in determining ouabain binding affinity of this isoform among different species (Price and Lingrel, 1988; Cantley et al., 1992; Jaisser et al., 1992). Using site-directed mutagenesis techniques, several laboratories have now modified the binding affinity of functional NaK-ATPase in transfected cells. The obvious criticism of NaK-ATPase as a “receptor” is that any circulating digitalis-like factor would indiscriminantly inhibit sodium pump function in all cells. This can be rebutted in part by a-isoform subunit selectivity, as noted above. It is also possible that physiological release of a digitalis-like factor would occur selectively as an autocrine or paracrine autocoid. reaching concentrations sufficient to inhibit NaK-ATPase within a restricted area in a given tissue or organ. Higher levels or unrestricted release of such a factor sufficient to induce hypertension could occur only under pathophysiological conditions. How-

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ever, this wide variation in ouabain binding affinities among a-subunit isoforms and in the same isoform among species seems inconsistent with the hypothesis that the cardiac glycoside binding motif on NaK-ATPase serves as a receptor for an endogenous ligand that regulates active cation transport by this enzyme. Nevertheless, the varying affinities of the asubunit isoforms for the cardiac glycosides would provide the basis for some cellular selectivity for an endogenous cardiac glycoside-like hormone. As previously articulated (Kelly and Smith, 1989), we believe that the key arguments in support of the existence of an endogenous form of digitalis based on the “NaK-ATPase as receptor” hypothesis rest on inappropriate analogies and unproven assumptions, despite the fact that such a circulating inhibitor provides a convenient, inclusive explanation for a number of pathophysiological phenomena. The existence of an endogenous ligand for the digitalis binding site acting as a physiological regulator of plasmalemmal cation flux also seems to run counter to a number of the known design criteria for regulating signal transduction in eukaryotic cells. Hormones, whether autocrine, juxtacrine, paracrine, or endocrine, are recognized by specific receptor proteins, either on the cell membrane or intracellular in the case of most steroid hormone receptors, that subsequently result in the generation of downstream signal transduction events. Whatever intracellular signal results, it is always subject to modulation that often leads to amplification of the original signal, and subsequently to the initiation of mechanisms that depress the cell’s responsiveness to subsequent similar hormonal signals. As presently envisioned, the endogenous digitalis-like factor would act on an integral cell membrane protein, the NaK-ATPase, and directly affect transmembrane ion flux, a mechanism that is more analogous to certain drugs (e.g., dihydropyridine Ca2+ channel blockers) and toxins (e.g., a-bungarotoxin). Some neurotransmitters do act by inducing conformational changes in integral membrane proteins, thereby directly regulating plasmalemmal ion flux, but in each case their molecular “receptor” is located in close apposition to a synaptic junction at the site of neurotransmitter release that is limited to restricted plasmalemmal domains of specific neurons in specialized areas of the nervous system. Typically, high concentrations of an appropriate degradative enzyme are close at hand to limit diffusion of the neurotransmitter away from its target, and efficient specific mechanisms exist to facilitate reuptake and inactivation of the neurotransmitter. Finally, the affinity of the target protein for the neurotransmitter, once bound, is often rapidly altered by changes in membrane potential or other mechanisms to facilitate release of the signaling factor.

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Thus, the mechanism of action of a putative endogenous regulator of NaK-ATPase that structurally resembles the cardiac glycosides does not appear to resemble canonical plasmalemmal signal transduction pathways. A number of endogenous compounds that regulate the activity of the sodium pump have been described, however, including catecholamines, insulin, thyroxine, mineralocorticoids, and other hormones and autacoids. Most of these compounds affect the sodium pump indirectly by varying the intracellular sodium concentration, directly by increasing the number of pump sites in the membrane, or by phosphorylation of the enzyme (Eisner and Smith, 1992; Ikeda et a/., 1991; McGill, 1991; Ibarra et a / . , 1993; Kamitani e f al., 1992; Chibalin et al., 1992). In addition, inorganic compounds such as vanadate, acting as a pyrophosphate analog within the cell, can inhibit phosphorylation of several cation-transporting enzymes, including NaK-ATPase. However, none of these hormones or inorganic compounds has been shown to bind to the cardiac glycoside binding site.

IV. Criteria for Identifying Digitalis-like Factor(s)

A. General Approach All of the theoretical objections noted above become moot if an endogenous ligand for the cardiac glycoside binding site can be isolated and characterized and shown to be present in physiologically relevant concentrations either locally within a specific tissue or circulating in plasma. The criteria traditionally used for identifying endogenous digitalis-like factors include (1) selectivity for NaK-ATPase; (2) inhibition of NaK-ATPase activity; (3) competition for binding with radiolabeled cardiac glycosides and inhibition of binding by [K+],; (4) inhibition of ouabainsensitive sodium pump activity in intact cells or tissues (and, as a corollary, a positive inotropic effect in cardiac muscle preparations); and ( 5 ) competition with authentic cardiac glycosides for binding sites on cardiac glycoside-specific antibodies. The last criterion, usually labeled “digoxin-like immunoreactivity” based on the widespread availability of polyclonal and monoclonal antidigoxin antibodies for clinical use, has been the most controversial and least reliable (Goto et al., 1991). This would be expected, given the highly variable and unpredictable affinity of other authentic cardiac glycosides to antibody preparations raised against digoxin as a hapten. Additional criteria that could be used to validate a candidate compound as “cardiac glycoside-like” include bioassays for sodium pump inhibition in a variety of cell types (Anderson et al., 1991) and physical-chemical

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approaches that, while rarely used, can be more specific than those listed above. These include high-resolution chromatographic, nuclear magnetic resonance (NMR), and mass spectroscopic techniques. For a comprehensive and thoughtful analysis of the techniques used by many investigators to identify endogenous digitalis-like factors, including the pitfalls associated with immunological and purified enzyme and radioreceptor assays as well as bioassay methods, the reader is referred to a recent review by Goto et al. (1992). The remainder of this review examines evidence purporting to support the existence of endogenous ligands for the cardiac glycoside binding site, emphasizingreports that have appeared since 1990.

B. Digoxin-like Immunoreactivity Despite its limitations, digoxin-like immunoreactivity,often in conjunction with other approaches, continues to be used to define patient populations that may have increased levels of digitalis-like factors. Naomi et al. (1991) suggested using antidigoxin antisera with differing affinities for authentic digoxin to establish a profile of immunoreactivity that could be used to track the presence of endogenous digitalis-like factors among patients with a variety of clinical conditions, an approach they termed establishing an “immunochemical footprint” of the endogenous compound. Graves and colleagues (Graves et al., 1984; Seely et al., 1992)have also combined digoxin radioimmunoassay with other hormonal indices of volume status to track digoxin-likeimmunoreactivityin women with hypertension associated with pregnancy. These authors were among the first to note the association between pregnancy and increased levels of digoxin-like immunoreactive factors in plasma, and their recent evidence suggests that plasma levels increase in hypertensive pregnant women, particularly those with proteinuria (i.e., preeclampsia) (Seely el al., 1992). There was no difference in sodium pump activity in washed erythrocytes from normotensive or hyertensive pregnant women. No attempt was made in this study to examine the effects of plasma or plasma extracts from preeclamptic patients on sodium pump function of erythrocytes from normotensive pregnant women, despite the differences in plasma levels of digoxin-like immunoreactivity. In a recent report Graves et al. (1993) also examined end-stage renal failure patients on peritoneal dialysis. They identified a single fraction in the dialysates of these patients, following ultrafiltration and high-performance liquid chromatography (HPLC), that had digitalislike NaK-ATPase inhibitory activity. Levels of this factor in dialysate correlated positively with NaK-ATPase inhibitory activity in serum (r = 0.64) and with the degree of weight gain (and therefore, presumably, volume expansion) between dialysis procedures (r = 0.67). Neither the

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digitalis-like activity in serum or the dialysate had physical-chemical characteristics similar to those of authentic ouabain or digoxin (or a number of other cardenolides and steroids), as judged by retention time on analytical reversed phase HPLC. Miyagi et al. (1991) have also examined “ouabain-like” NaK-ATPase inhibitory activity in the plasma of patients with pregnancy-induced hypertension. As in the report by Seely et al. (1992), they found no difference in erythrocyte sodium pump activity, at least as judged by NaK-ATPase activity by erythrocyte ghosts among patient and control populations. While they did not examine digoxin-like immunoreactivity in plasma per se, plasma extracts containing NaK-ATPase inhibitory activity were unaffected by polyclonal antidigoxigenin Fab fragments at concentrations sufficient to remove the pharmacological activity of up to 30 nM authentic digoxin. Interestingly, this antidigoxigenin Fab preparation had little effect on the NaK-ATPase inhibitory activity in their bioassay of 30 nMauthentic ouabain. For this reason, and because other known inorganic inhibitors of NaK-ATPase (e.g., vanadate) could be excluded, these authors described their NaK-ATPase activity as being “ouabain-like” without any further evidence bearing specifically on this point. Interestingly, “ouabain-like” NaK-ATPase inhibitory activity was elevated both in patients with pregnancy-induced hypertension and in a group of normotensive nonpregnant women compared to pregnant women with normal blood pressure. Miyagi et al. (1991) suggested that the normal physiological response in pregnancy might be to reduce plasma levels of an ouabain-like factor as volume expansion occurs, and that levels that remain inappropriately within the normal range contribute to the development of hypertension (Miyagi et al., 1991). Several other reports have also appeared that examine the relationship between plasma levels of digoxin-like immunoreactivity and the development of hypertension in pregnancy (Wolfe et al., 1990; Kaminski and Rechberger, 1991). Valdes and co-workers (Shaikh et al., 1991) have published a comprehensive evaluation of digoxin-like immunoreactivity in serum and tissue samples from several mammalian species, including humans, using a commercially available source of polyclonal antidigoxin antibodies that has been widely used by these and other investigators in the literature. The highest concentrations of digoxin-like immunoreactivity were present in adrenal extracts prepared from rat and human sources compared with other tissues or with serum samples. To determine whether increased secretion of digoxin-like immunoreactive factors could be detected from the adrenal glands, serum samples were obtained from the infrarenal inferior venae cavae of anesthetized nephrectomized dogs and from lumbar veins collecting adrenal drainage. There was a 3-fold increase in digoxin-

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like immunoreactivity in serum from the lumbar veins compared to mixed infrarenal venal caval serum. Using bovine adrenal cortex as a source of digoxin-like immunoreactivity , these researchers demonstrated that, following preparative as well as analytical HPLC, digoxin-like immunoreactivity coeluted with an authentic digoxin standard. Although the authors reported preliminary evidence from mass spectroscopic studies suggesting that the principal digoxin-like immunoreactive species obtained from bovine adrenal glands is structurally very similar to digoxin, no definitive identification was provided in this report. Digoxin-like immunoreactivity in plasma samples from neonates has also been the subject of numerous reports. Potentially toxic levels of digoxin have been reported in infants who have not received the drug. This has obvious implications for therapeutic drug monitoring of digoxin therapy in young children, as well as suggesting an important limitation for the use of digoxin radioimmunoassays (RIAs) in the forensic toxicology of suspected digoxin poisoning in infants. The limitations of a number of immunoassay techniques and reagents widely used in therapeutic drug monitoring by clinical laboratories have been recently examined by several groups (Stone et al., 1990; Morris et al., 1990, Liu er al., 1990b). Immunoassay techniques using fluorescence polarization rather than radiolabeled compounds were generally no better than standard RIA techniques and may show increased levels of “digoxin-like” immunoreactive compounds, depending on the agent used to precipitate serum proteins (Mojaverian et al., 1989). As might have been predicted from experience with immunoassays using polyclonal antisera, monoclonal antibodies developed against authentic digoxin have been reported to exhibit a range of cross-reactivities with digoxin-like immunoreactivity in cord blood, including several that exhibited no detectable binding (Wahyono et al., 1991; Loucari-Yiannakou et al., 1990; Terano et al., 1991; Engel and Khanna, 1992). While the availability of high-affinity digoxin-specificantibodies may eliminate falsepositive digoxin immunoassays in therapeutic drug monitoring of neonates and patients with other clinical conditions such as uremia, characterized by substances that tend to interfere with the assay (not all of which are truly immunologically cross-reactive compounds), more cumbersome but selective analytical techniques, such as HPLC followed by RIA, may be necessary to exclude such interfering substances. The chemical identities of all of the factors responsible for digoxin-like immunoreactivity in cord blood, placenta, and amniotic fluid remain unclear, but almost certainly include sulfated and nonsulfated steroids (Chasalow and Blethen, 1990; Yun et al., 1992). In most reports, however, not all digoxin-likeimmunoreactivity in cord blood could be accounted for by steroid compounds known

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to cross-react to a limited extent with antidigoxin antibodies, leaving the possibility that some fraction of the remaining signal represented a physiological regulator of NaK-ATPase (Seccombe et al., 1989; Chasalow and Blethen, 1990). Aside from its presence in cord blood, amniotic fluid, and the plasma of pregnant women, digoxin-like immunoreactivity has been studied in other contexts, with or without concomitant measurements of NaKATPase inhibitory activity. These reports have examined the relationship between cardiac glycoside-like activity and sodium excretion and/or blood pressure in patients or experimental animals (Takahashi et al., 1991; Morise et al., 1989; Nakagawa et al., 1990; Szylman et al., 1990; CastanedaHernandez, 1989; Vargas et al., 1991; Liu et al., 1990a; Longerich et af., 1990). Interestingly, two reports indicated that plasma NaK-ATPase inhibitory activity, alone (Delva et al., 1990) or coupled with digoxin-like immunoreactivity (Bagrov et al., 1991), increases in patients following myocardial infarction or immediately following transient coronary artery occlusion following percutaneous transluminal coronary angioplasty. These authors speculated that the increased digoxin-like immunoreactivity could reflect increased release of an endogenous agonist with cardiac glycoside-like properties. Bagrov et al. (1991) also noted a dissociation between changes in plasma levels of digoxin-like immunoreactivity and NaK-ATPase inhibitory activity. No attempt was made to characterize further the chemical structure of the factors in these reports. Shilo et al. (1989) have reported that an endogenous digoxin-like factor, isolated from urine, protected rats against lethal arrhythmias induced by authentic digoxin when infused intraarterially with the drug. Interestingly, Agbanyo, Khatter, and colleagues have partially characterized an extract from pig heart with positive inotropic activity and other cardiac glycoside-likeproperties, including digoxin-like immunoreactivity (Agbanyo and Khatter, 1990; Navaratnam et al., 1990; Khatter et al., 1991). Their tissue extract inhibited NaK-ATPase preparations obtained from cardiac sarcolemmal membranes as well as commercially available canine kidney cortex enzyme, blocked %Rbuptake into adult rat ventricular slices, and appeared to cross-react with antidigoxin antibodies. This same tissue extract also increased developed force in right ventricular trabeculae, without evidence of toxic contracture, even at higher driving frequencies. These authors excluded catecholamines and inorganic compounds as potentially contributing to the biological activity they had isolated, but did not report any further characterization of their activity. Doris, Stocco, and colleagues have published three reports demonstrating the release from adrenaf cortex and/or a transformed murine adrenocortical cell line, of a factor(s) with digoxin-like immunoreactivity into

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serum-free medium (Doris et al., 1989; Doris and Stocco, 1989; Doris, 1992).HPLC fractions of this medium exhibitingdigoxin-likeimmunoreactivity also inhibited [3H]ouabainbinding to erythrocyte membranes. Interestingly, the addition of one of several inhibitors of steroid hormone synthesis from cholesterol had no effect on the rate of appearance of digoxin-like immunoreactivity from either adrenal tissue or adrenocortical tumor cells (Doris et af., 1989). Finally, Goto et af. (1990) have reported the isolation of a compound from human urine that cross-reacted with severaldigoxin-specificantibody preparations and inhibited 86Rbuptake into human erythrocytes. These authors went substantially further than most reports by providing data from a number of physical chemistry analyses, including proton NMR and fast atom bombardment (FAB) mass spectroscopy following repeated chromatographic separations. The material was “indistinguishable” from authentic digoxin. These authors could not exclude possible contamination of their urine samples by subjects exposed to authentic digoxin, although this appeared to be unlikely. The authors also identified a more polar digoxin-like immunoreactive factor from human urine, or ‘‘ouabaindisplacing compound” (i.e., ODC-1) that, unlike authentic ouabain, induced large vessel (bovine pulmonary artery) endothelium to increase its rate of release of the vasoconstriction autacoid endothelin into (serumfree) culture medium (Yamada et af., 1990). The presence of other known endothelin secretagogues within the ODC-1 fractions was not systemically excluded. Nevertheless, these results, if confirmed,would provide another mechanism by which an ouabain-likefactor could contribute to an increase in peripheral vascular resistance and to the pathogenesis of hypertension.

V. Identification of Digitalis-like Factors: Recent Developments

A. Candidate Compounds A number of endogenous compounds with NaK-ATPase inhibitory activity have been identified, including steroids and amphipathic lipids. Although each of these compounds could test positive in one or more of the standard immunoassay and/or bioassay or biochemical tests for cardiac glycosidelike activity, none was capable of high-affinity binding to digoxin-specific antibodies and most presumably inhibited NaK-ATPase activity by a mechanism@)unrelated to specific binding to the ouabain binding site on the enzyme. Lichstein et al. (1991) isolated three digitalis-like compounds from bovine plasma and conclusively identified one as 11,13-dihydroxy1Coctadecaenoic acid, which they suggested may represent at least a

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portion of the cardiac glycoside-like biological activity in extracts of bovine adrenal glands originally reported by Tamura et d.(1988). Sohn et d. (1992) examined the ultrafiltrate obtained from uremic patients undergoing hemodialysis and identified fractions following gel filtration that inhibited activity of isolated NaK-ATPase and &Rb transport in aortic strips. The active fractions did not bind to antidigoxin antibodies. Interestingly, there has been one recent report of artifactual digoxin-like immunoreactivity in dialysis ultrafiltrate, apparently attributable to a plasticizer that tended to leach from hemodialysis tubing (Malik et al., 1991). A number of other approaches have been used in the search for the identity of endogenous digitalis-likefactors, although most recent comprehensive approaches to this problem have used analytical separation procedures, followed by one or more physical-chemical analysis techniques. This is essential given the lack of specificity of many of the immunological, radioreceptor, enzyme inhibition, and ion transport assays. Nevertheless, subtle but revealing differences between authentic cardiac glycosides and endogenous compounds that meet many of the criteria for being “digitalislike” can be identified. One such compound that may have important physiological effects is the endogenous NaK-ATPase inhibitor isolated from hypothalamic tissue originally described by Haupert and Sancho (1979). Haupert and colleagues have gone on to show that this factor is specific for NaK-ATPase, inhibits the enzyme reversibly and with high affinity (Haupert et al., 1984; Anner er al., 1990), and can prevent ouabain binding, although, unlike ouabain, it does not facilitate phosphorylation of the enzyme by inorganic phosphorus. In a recent report Haupert and colleagues examined the mechanism by which bovine hypothalamic inhibitory factor (HIF) could affect vasomotor control of aortic and pulmonary arterial rings obtained from SHR and normotensive Sprague-Dawley rats (Janssens er al., 1993). HIF induced a pronounced vasoconstriction of SHR pulmonary artery rings that was dose dependent and reversible and that was significantly greater than in pulmonary arterial rings from control animals or in aortic rings from either SHR or control rats. This effect of HIF could be completely prevented by M phentolamine. Curiously, M did not induce vasoconouabain itself at concentrations as high as striction of pulmonary artery rings, which these authors suggested may be due to a lower affinity of aortic NaK-ATPase for authentic ouabain compared to HIF. The facilitation of sympathetic neurotransmitter release and inhibition of presynaptic neuronal reuptake with digitalis glycosides has been known for some time (Hougen et al., 1981; Gillis and Quest, 1979) and has been proposed as a mechanism by which an endogenous digitalis-like factor could influence peripheral vascular resistance. These data from Janssens et al. (1993) highlight the fact that pharmacological

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responses, such as vasoconstriction, to cardiac glycosides or their putative endogenous congeners in bioassay preparations may be due to any of several mechanisms.

B. Endogenous Ouabain 1 . Identification of Endogenous Ouabain in Human Plasma In a series of recent manuscripts, Hamlyn, Ludens, and colleagues described the isolation and characterization of cardiac glycoside-like activity from human plasma (Hamlyn et al., 1989, 1991; Harris et al., 1991; Bova et al., 1991; Ludens et al., 1991; Mathews et al., 1991). This activity was initially isolated from over 80 liters of human plasma, acquired from patients undergoing routine plasmapheresis, who had not been prescribed cardiac glycosides for therapeutic reasons. Following preparative ion exchange and reversed phase chromatography, specific fractions were exposed to a molar excess of partially purified lamb kidney NaK-ATPase under conditions that favored cardiac glycoside binding (i.e., batch enzyme affinity extraction). After washing, the NaK-ATPase was exposed to buffers containing EDTA, which would favor dissociation of ligands bound to the ouabain binding site. The resulting supernatant was filtered and subjected to sequential chromatographic separation steps, and a single peak containing cardiac glycoside-like activity was identified, as assayed by &Rb flux into human erythrocytes, [3H]ouabainbinding to canine kidney NaK-ATPase, and inhibition of NaK-ATPase activity by a coupled optical assay technique (Hamlyn et al., 1982). Of note, digoxin-likeimmunoreactivity using standard antidigoxin polyclonal antisera did not track closely with a plasma fraction(s) exhibiting cardiac glycoside-like activity, as defined by the other assay procedures noted above, and therefore was not used as a criterion for screening purposes. A protonated molecular ion was identified in an HPLC fraction containing cardiac glycoside-like activity by FAB mass spectroscopy, with an m/z of 585.2, identical to the calculated value for authentic ouabain (Mathews et al., 1991). Tandem mass spectroscopy of the protonated molecular ions of authentic ouabain and the fraction containing cardiac glycoside-like activity yielded identical daughter ion spectra, including major fragments with an m/z of 439.2, as predicted for the aglycone form of ouabain. Additional physical-chemical analytical techniques, such as resolution by chromatographic separation columns capable of resolving, for example, stereoisomers of ouabain, yielded identical retention times for both authentic ouabain and an absorbance peak corresponding to the fraction(s) containing cardiac glycoside-likeactivity derived from plasma. A rabbit polyclonal antiserum raised to ovalbumin-conjugated authentic

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ouabain bound the cardiac glycoside-like activity with an affinity similar to that of authentic ouabain (Hams et al., 1991). The source of an endogenous ouabain-like compound (OLC) appeared to be the adrenal glands, as the highest tissue levels of OLC were present in (rat) adrenal glands and plasma levels declined in adrenalectomized animals, as assayed by a ouabain antibody enzyme-linked immunosorbent assay (ELISA). Cultured adrenocortical cells obtained from bovine adrenal glands also released ouabain-like activity, as detected by ELISA, into serum-free medium (Hamlyn et a / . , 1991).

2. Ouabain as a Circulating Natriuretic Hormone In subsequent reports Hamlyn and colleagues have presented evidence that plasma levels of the OLC, although apparently increased in some experimental models of hypertension that are characterized, at least initially, by volume expansion and sodium retention (e.g., the DOCA salt model in uninephrectomized rats given deoxycorticosterone acetate and 0.9% saline to drink) (Hamlyn et al., 1991), did not change in dogs subjected to either acute volume expansion or acute hemorrhage (Boulanger et al., 1993). The release of ouabain-like activity across the adrenal circulation also did not change during volume expansion or hemorrhage, but remained constant at a ratio of approximately 5 : 1 (i.e., adrenal venous:adrenal arteriaI ouabain-like activity), although, as expected, both cortisol and aldosterone release increased following hemorrhage. Inierestingly. in separate experiments using an anesthetized dog model, although again volume expansion did not affect plasma ouabain-like activity as measured by ELISA using ouabain-specific antibodies, NaKATPase inhibitory activity in desalted plasma extracts did increase, as detected by a coupled optical assay linked to NaK-ATPase activity (Ludens et al., 1993). These data would suggest that endogenous ouabain, as detected by the ouabain antibody-based ELISA, is distinct from other cardiac glycoside-like activity in plasma. The latter may represent a separate specific inhibitor of NaK-ATPase or, more likely, reflect the combined effects of a number of nonspecific endogenous compounds on the isolated NaK-ATPase in the coupled optical enzyme assay. Whether a link exists between an ouabain-like factor and hypertension therefore remains inconclusive. It is now clear, however, that chronic administration of a cardiac glycoside in doses somewhat higher than those used therapeutically in humans can induce hypertension in experimental animals, even in a “digitalis-resistant” species such as the rat. However, at least a 50% reduction in renal mass, in addition to repeated intraperitoneal injections of the drug, appears to be necessary to induce a sustained rise in blood pressure (Yamada et al., 1992; Yuan et al., 1993).

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3. Ouabain as a Centrally Acting Autacoid Huang, Leenen, and colleagues (Huang and Leenen, 992; Huang et al., 1992), expanding on earlier work by Takahashi et a1 (1984, 1987), also have implicated a role in the etiology of experiment 1 hypertension for an endogenous OLC in the brain. Administration of either authentic ouabain or ouabain-like activity prepared from rat hypothalamus, when injected directly into the cerebral ventricles of rats, increased blood pressure, heart rate, and renal sympathetic nerve activity, an effect that could be mimicked by the intracerebroventricular infusion of 0.3 M NaCl. Fab fragments of antidigoxin antibodies,given in concentrationsthat prevented the central actions of authentic ouabain, also prevented the central sympathoexcitatory effect of intracerebroventricular hypertonic saline, while a non-Fab antibody control (y-globulin) was ineffective. K. Yamada et al. (1992) have used a similar approach, studying the effects of Fab fragments on natriuresis as well as blood pressure following intracerebroventricular injections of hypertonic saline. Antidigoxin antibodies have also been used in immunohistochemicalstudies to identify endogenous digitalis-like factors in the periventricular regions of canine and primate brain tissue (H. Yamada et al., 1992). Leenen et al. (1993) have also examined the effects of high dietary sodium on ouabain-like activity in plasma, peripheral organs, and the pituitary gland and the hypothalamus of SHR and WKY rats. As assayed by measuring ATP hydrolysis by canine kidney cortex NaK-ATPase, ouabain-like activity was detected in extracts prepared by tissue homogenization followed by centrifugation and preparative chromatography on disposable C,8 cartridges. Adrenal and hypothalamic ouabain-like activity content was higher in SHR than WKY rats on control and high-sodium diets. On a high-sodium diet ouabain-like activity increased in peripheral organs and in the pituitary gland and the hypothalamus of both SHR and WKY rats, but it was higher in SHR rats. In a recent preliminary report Leenen and Huang have demonstrated a reduced central sympathoexcitatory response to intracerebroventricular ouabain in hypertensive Dahl salt-sensitive rats fed a high-salt diet compared to normotensive Dahl salt-sensitive rats on a low-salt diet or to Dahl salt-resistant animals on either diet (Leenen and Huang, 1993), an observation they attributed to what they regard as a probable increase in endogenous ouabain release centrally and therefore greater receptor occupancy. In another model of genetic hypertension, Ou et al. (1993) quantified the V,, of NaK-ATPase and the KDof the enzyme for authentic ouabain for both low- and high-affinity ouabain binding sites in rat hypothalamic synaptosomes prepared from SHR and WKY animals on highand low-sodium diets. As expected, blood pressure was significantly

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higher in SHR than WKY rats, and a high-salt diet accentuated the degree of hypertension in the SHR animals. The total NaK-ATPase V,, was higher in SHR than WKY rats on either diet, and on high salt a third NaKATPase isoenzyme highly sensitive to inhibition by ouabain appeared in synaptosomes prepared from SHR hypothalami. As this would tend to favor increased inhibition of sodium pump activity by a hypothalamic factor with ouabain-like binding kinetics, it could explain the higher blood pressures observed in SHR rats fed a high-salt diet. These data may provide a link between the identification of ouabain as a candidate endogenous cardiac glycoside with an extensive older literature that has pointed to the importance of penventricular tissue in the anteroventral third ventricular (i.e., AV3V) region of the brain in the elaboration of a digitalis-like factor(s) that might act locally to induce or sustain hypertension in experimental animal models (Buggy er af., 1984; Berecek et af., 1983; Songu-Mize et af., 1982; Takahashi et af., 1984).

4. Endogenous Ouabain in Congestive Heart Failure A clinical condition typically characterized by volume expansion is congestive heart failure, in which elevated levels of a cardiac glycoside-like natriuretic factor have been postulated. Gottlieb et af. (1992) examined ouabain-like activity, detected in desalted plasma extracts, using a polyclonal anti-ouabain antibody-based ELISA in 51 patients, most of whom had moderate to severe (i.e., New York Heart Association functional class 111 and IV) congestive failure. Claiming limited cross-reactivity of their antibody preparations with digoxin, these authors asserted that ouabain-like activity also could be evaluated in patients who were receiving this cardiac glycoside. They reported no difference in ouabain-like activity as detected in plasma by ouabain-specific ELISA, whether or not patients were receiving digoxin. The average plasma ouabain concentration in normal controls was 0.44 k 0.20 nM (mean 2 SD; range, 0.16-0.77 nM), 1.59 +- 2.2 nM (range, 0.17-8.76 nM) in heart failure patients receiving digoxin, and 1.52 +- 2.58 nM in patients not on maintenance digoxin, a significant difference between patients and controls. Although all control subjects had ouabain levels of less than 1 nM, patient ouabain activity ranged as high as 8 nM, a concentration of cardiac glycoside usually only achieved by patients after major overdose, yet these patients were apparently without clinical symptoms or signs of toxicity. Plasma ouabain levels did not correlate with volume status, at least as reflected by left ventricular filling pressure and by right atrial pressure, nor was there any significant correlation with the glomerular filtration rate, although there

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was a weak negative correlation between the log [ouabain] and cardiac index (r = -0.62) or mean arterial pressure (r = -0.51) among 19 of the patients studied. The authors concluded that although there was no support in these data for a role for an endogenous ouabain in the regulation of sodium or volume homeostasis, the statistically significant negative correlations with cardiac index and mean arterial pressure implicated endogenous ouabain in having a possible role in maintaining the circulation in patients with congestive heart failure. It should be noted that in their original identification of the pharmacological properties of ouabain-like activity in human plasma, Hamlyn et af. (1991) did demonstrate a doseand time-dependent increase in developed force of isolated guinea pig atria with kinetics similar to that of authentic ouabain, suggesting that endogenous ouabain might be released into the circulation in order to support cardiac function. In two other studies (Delva et al., 1991; Liu et al., 1990) of cardiac glycoside-like activity in the plasma of patients with congestive heart failure, plasma levels of NaK-ATPase inhibitory activity in plasma extracts deproteinized by boiling and of digoxin-like immunoreactivity were studied in patients compared to control subjects. Each of these values declined after short-term therapy with dobutamine (in the study by Liu et af., 199Oa). As in the study by Gottlieb et al. (1992), Delva et al. (1991) were able to demonstrate a weak but significant negative correlation between the ability of plasma to inhibit NaK-ATPase and cardiac index (r = 0.49) and a positive correlation (r = 0.50) between plasma digoxin-like immunoreactivity and NaK-ATPase inhibitory activity. Also relevant is the fact that the NaK-ATPase inhibitory activity isolated from bovine hypothalamus by Hallaq and Haupert (1989) (i.e., HIF) also has been shown to increase the amplitude of cell shortening in spontaneously beating isolated neonatal rat heart cells, accompanied by an increase in intracellular calcium, although it is not known whether plasma levels of this NaK-ATPase inhibitor are elevated in patients with congestive heart failure.

5 . Endogenous Ouabain in Genetic Models

of Hypertension

In a prodigious effort Ferrandi et al. (1992), using methods based on those developed by Haupert and colleagues for isolating HIF (Haupert and Sancho, 1979; Haupert et af., 1984), isolated ouabain-like factor activities from the hypothalamus as well as the adrenals of Milan hypertensive strain (MHS) and Milan normotensive strain (MNS) rats. They tested the ability of these hypothalamic and adrenal extracts to inhibit canine kidney

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cortex NaK-ATPase by coupled-enzyme assays, to inhibit 32P-labeledATP hydrolysis and to displace [3H]ouabainfrom NaK-ATPase, as well as to inhibit %Rbuptake into erythrocytes obtained from normotensive human donors. Preparations of ouabain-like activity in extracts from rat hypothalami required 200-300 animals from each strain for each individual purification. They showed conclusively that the ouabain-like activity isolated from rat hypothalamus was similar, if not identical, to HIF obtained originally from bovine brain. Using a separation procedure similar to that used for isolating ouabain-like activity from the hypothalamus, an active fraction was also obtained from adrenal tissue with characteristics that were functionally identical in the bioassay and biochemical assays listed above to the hypothalamic fractions. It is instructive to note that both MHS and MNS animals were housed under identical conditions and ate the same chow, yet hypothalami isolated from MHS rats contained significantly more HIF activity, suggesting, but not proving, that the NaKATPase inhibitory activity may have been synthesized de n o w in these animals. Interestingly, this hypothalamic/adrenal activity was not “ouabainlike” in several respects. Most notably, while authentic ouabain exhibits a 100- to 1000-fold lower affinity for the predominant a-subunit isoform in rat renal tubular NaK-ATPase preparztions than the predominant a-subunit isoform in rat brain synaptosomes, the estimated EC,, for binding to NaK-ATPase in either preparation by the hypothalamic/ adrenal ouabain-like activity differed only by a factor of 5. The affinity of the rat brain HIF-like activity also had a more pronounced inhibitory action in rat renal tubular NaK-ATPase obtained from adult MHS or MNS animals compared to the enzyme isolated from young prehypertensive animals, while there was no kinetic distinction for ouabain inhibition of enzyme isolated from either young or old animals. Although the recovery of ouabain-like or HIF-like activity from hypothalamic tissue of MHS and MNS animals is difficult to quantitate, Ferrandi et al. (1992) felt that the data, whether normalized to grams of tissue or to numbers of animals sacrificedfor each purification, clearly indicate that hypothalamus from MHS rats contains approximately 5-10 times the inhibitory activity present in hypothalamus from their normotensive controls. Haupert and colleagues have also reported that the mechanism by which HIF interacts with NaK-ATPase differs from that of ouabain and other authentic cardiac glycosides (Haupert er al., 1984; Anner et af., 1990),and further chemical characterization of this activity, while not yet complete, apparently indicates that HIF is structurally different from ouabain (Tymiak et al., 1993).

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VI. Is Ouabain the Endogenous Digitalis? In our view none of the evidence presented in these reports establishes unequivocally that an endogenous cardiac glycoside-like compound exists as a physiological regulator of NaK-ATPase activity. For example, in the study by Gottlieb et al. (1992) of ouabain-like activity in congestive heart failure, plasma levels of ouabain were quantified by ouabain-like immunoreactivity, not by the series of more rigorous chemical separation and analytical techniques used by Hamlyn and colleagues in their original reports (Hamlyn et al., 1991; Ludens ef al., 1991; Mathews et al., 1991). We feel that until there is independent verification of the existence of ouabain as a physiological regulator, it is inappropriateto rely on immunoreactivity alone as an index of ouabain-likeactivity, even though individual patient samples were treated to remove some plasma components that could have interfered with the ELISA assay (Kelly and Smith, 1992). Earlier experiments by one of us (Selden et al., 1974; Selden and Smith, 1972), designed to examine the pharmacokinetics of ouabain using a wellcharacterized anti-ouabain polyclonal antisera, did not detect any evidence of endogenous ouabain-like activity in plasma or urine. While it is possible that ouabain can be isolated and positively identified from plasma following serial preparative and analytical chromatographic steps, it is very difficult to exclude contamination by authentic cardiac glycosides, including trace contaminants of plant-derived cardenolides in the diet. In addition, the experiments described above by Huang and Leenen (1992), in which intracerebroventricular injection of Fab fragments of digoxin-specific antibodies reversed the sympathoexcitatory effects of concomitantly administered hypertensive saline, presumably by binding to an endogenous ouabain-like agent-although provocative-are unconvincing. No chemical characterization of the ouabain-like activity in their hypothalamic and pituitary extracts has been published, and perhaps more importantly for the analysis of the data in this report, the intracerebroventricular injection of y-globulin was not an adequate control for the Fab antidigoxin antibody preparation. The reports by Leenen and colleagues (Huang and Leenen, 1992; Huang et al., 1992; Leenen et al., 1993) that describe cardiac glycoside-likeactivity in tissue or plasma samples do not include an appropriate validation of their assay procedure(s) to exclude artifactual signals, a criticism that can be made of many of the studies reported in this review. Standard curves for authentic cardiac glycosides should always be prepared in buffers, solvents, or “tissue extracts” identical to samples containing unknown quantities of putative endogenous digitalis-like factors. While this may be complicated, at least in theory,

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by detectable basal levels of digitalis-like activity in plasma or tissue extracts, one of several strategies can be used to selectively remove this activity, such as the batch enzyme affinity extraction technique of Hamlyn e? a / . (1991). Again, appropriate controls must be provided to account for nonspecific adsorption of compounds present in tissue or plasma extracts by the (relatively impure) NaK-ATPase preparation. Given the many false starts and blind alleys that have characterized research in this field over the past two decades, it would seem prudent to insist that rigorous analytical techniques and controls be applied to all studies in humans and experimental animals. Furthermore, if an adrenaland/or brain-derived ouabain-like autacoid exists, as suggested by the work of Hamlyn ef al. (1991), Haupert (19881, Ferrandi et al. (1992), Huang and Leenen (1992), Tamura et al. (19881, Doris (1992), and others, then ouabain should be identifiable in defined medium conditioned by primary cultures of adrenocortical or hypothalamic cells (or representative cell lines). Net secretion of ouabain over time, in excess of the cells’ basal ouabain content, would provide more substantive evidence of ouabain synthesis, as opposed to uptake and sequestration of cardenolides or their precursors from the diet. Additional problems posed by the ouabain-like identity of a putative endogenous NaK-ATPase inhibitor include their unconventional structure in the context of most mammalian steroid biochemistry. As mentioned briefly above, the cis fusion of the A-B and C-D rings is unlike that found in steroid hormones or even in most bile salts. To our knowledge, the endogenous presence of the deoxy-L-sugar rhamnose is also unprecedented, as is the unusual unsaturated lactone at C,,. To quote Hamlyn and Manunta (1992) in a recent review, “While the presence of one of the features in a mammalian compound is unexpected, the finding of all three in one molecule of putative origin is remarkable.” Nevertheless, if net synthesis of ouabain, or a structurally similar congener or aglycone, can be demonstrated in mammalian cells, then it becomes more difficult a priori to exclude a role for an endogenous ouabain in the regulation of sodium homeostasis and blood pressure. Although nature has, in the past, provided exceptions to the prevailing conventional wisdom in molecular pharmacology, these considerations. along with our reservations raised earlier in this review about the concept of NaK-ATPase as a “receptor,” constitute important caveats in our opinion. The concept that either a circulating or a locally acting cardiac glycoside-like hormone or autacoid will be identified and proven to have a physiologically relevant role requires substantial additional experimental support before it can be accepted as fact.

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Hamlyn, J. M., and Manunta, P. (1992). Ouabain, digitalis-like factors and hypertension. J. Hypertens. 10, S99-S 11 1. Hamlyn, J. M., Ringel, R., Schaeffer, J., Levinson, P. D., Hamilton, B. P., Kowarski, A. A., and Blaustein, M. P. (1982). Acirculatinginhibitorof(Na++K+)ATPaseassociated with essential hypertension. Nature (London) 300,650-652. Hamlyn, J. M., Harris, D. W., and Ludens, J. H. (1989). Digitalis-like activity in human plasma. Purification, affinity, and mechanism. J. Biol. Chem. 264, 7395-7404. Hamlyn, J. M., Blaustein, M. P., Bova, S., DuCharme, D. W., Harris, D. W., Mandel, F., Mathews, W. R., and Ludens, J. H. (1991). Identification and characterization of a ouabain-like compound from human plasma. Proc. Natl. Aad. Sci. U.S.A. 88,6259-6263. Harris, D. W., Clark, M. A., Fisher, J. F., Hamlyn, J. M., Kolbasa, K. P., Ludens, J. H., and DuChanne, D. W. (1991). Development of an immunoassay for endogenous digitalislike factor. Hypertension 17, 936-943. Haupert, G. T. (1988). Physiological inhibitors of Na,K-ATPase: Concept and status. In “The Na+,K+-Pump,Part B; Cellular Aspects,” pp. 297-320. Liss, New York. Haupert, G. T., and Sancho, J. M. (1979). Sodium transport inhibitor from bovine hypothalamus. Proc. Natl. Acad. Sci. U.S.A. 76, 4658-4660. Haupert, G. T., Jr., C a d i , C. T., andcantley, L. C. (1984). Hypothalamic sodium-transport inhibitor is a high affinity reversible inhibitor of Na+,K+-ATPase.Am. J. Physiol. 247, F919-F924. Hougen, T. J., Spicer, N., and Smith, T. W. (1981). Stimulation of monovalent cation active transport by low concentrations of cardiac glycosides: Role of catecholamines. J. Clin. Inuest. 68, 1207-1214. Huang, B. S., and Leenen, F. H. H. (1992). Brain ouabain and central effects of dietary sodium in spontaneously hypertensive rats. Circ. Res. 70,430-437. Huang, B. S., Harmsen, E., Yu,H., and Leenen, F. H. H. (1992). Brainouabain-likeactivity and the sympathoexcitatory and pressor effects of central sodium in rats. Circ. Res. 71, 1059-1066. Ibarra, F., Aperia, A., Svensson, L.-B., Eklof, A.-C., andGreengard, P. (1993).Bidirectional regulation of Na+,K+-ATPase activity by dopamine and an a-adrenergic agonist. Proc. Natl. Acad. Sci. U.S.A. 90, 21-24. Ikeda, U., Huyman, R., Smith, T. W., and Medford, R. M. (1991). Aldosterone-mediated regulation of Na+,K+-ATPase gene expression in adult and neonatal rat cardiocytes. J. Biol. Chem. 266, 12058-12066. Jaisser, F., Canessa, C. M., Horisberger, J.-D., and Rossier, B. C. (1992). Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The 0 subunit modulates potassium activation of the Na,K-pump. J. Biol. Chem. 267, 16895-16903. Janssens, S. P., Kachoris, C., Parker, W. L., Hales, C. A., and Haupert, G. T., Jr. (1993). Hypothalamic Na+,K+-ATPase inhibitor constricts pulmonary arteries of spontaneously hypertensive rats. J. Cardiouasc. Pharmacol. in press. Kaminski, K., and Rechberger, T. (1991). Concentration of digoxin-like immunoreactive substance in patients with preeclampsia and its relation to severity of pregnancy-induced hypertension. Am. J. Obstet. Gynecol. 165, 733-736. Kamitani, T., Ikeda, U., Muto, S., Kawakami, K., Nagano, K., Tsuruya, Y., Oguchi, A., Yamamoto, K., Ham, Y., Kojima, T., Medford, R. M., and Shimada, K. (1992). Regulation of Na,K-ATPase gene expession by thyroid hormone in rat cardiocytes. Circ. Res. 7 l , 1457-1464. Katz, A. 1. (1982). Renal NaK-ATPase: Its role in tubular sodium and potassium transport. Am. J . Physiol. 242, F207-F219. Kelly, R. A., and Smith, T. W. (1989). The search for the endogenous digitalis: An alternative hypothesis. Am. J. Physiol. 256, C937-C950.

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