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A wealth of evidence supports the role of tumor immunotherapy as a vital therapeutic option in cancer. In recent decades, accumulated studies have revealed the anticancer activities of natural products and their derivatives. Increasing interest has been driven toward finding novel potential modulators of tumor immunotherapy from natural products, a hot research topic worldwide. These works of research mainly focused on natural products, including polyphenols (e.g., curcumin, resveratrol), cardiotonic steroids (e.g., bufalin and digoxin), terpenoids (e.g., paclitaxel and artemisinins), and polysaccharide extracts (e.g., lentinan). Compelling data highlight that natural products have a promising future in tumor immunotherapy. Considering the importance and significance of this topic, we initially discussed the integrated research progress of natural products and their derivatives, including target T cells, macrophages, B cells, NKs, regulatory T cells, myeloid‐derived suppressor cells, inflammatory cytokines and chemokines, immunogenic cell death, and immune checkpoints. Furthermore, these natural compounds inactivate several key pathways, including NF‐κB, PI3K/Akt, MAPK, and JAK/STAT pathways. Here, we performed a deep generalization, analysis, and summarization of the previous achievements, recent progress, and the bottlenecks in the development of natural products as tumor immunotherapy. We expect this review to provide some insight for guiding future research., This review aims to summarize the integrated research progress of natural products (e.g. curcumin) on the tumor immunotherapy and key intracellular pathways.

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3- complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+(+)-antagonism and suggest a route to isoform specific CTSs.

Stimulus-dependent elevation of intracellular Ca2+ affects gene expression via well-documented calmodulin-mediated signaling pathways. Recently, we found that the addition of extra- and intracellular Ca2+ chelators increased, rather than decreased, the number of genes expressed, and that this is affected by the elevation of [Na+]i/[K+]i-ratio. This assumes the existence of a novel Na+i/K+i-mediated Ca2+i-independent mechanism of excitation-transcription coupling. To identify upstream Na+i/K+i-sensitive genes, we examined the kinetics of transcriptomic changes in human umbilical vein endothelial cells (HUVEC) subjected to Na,K-ATPase inhibition by ouabain or K+-free medium. According to our data, microRNAs, transcription factors, and proteins involved in immune response and inflammation might be considered as key components of Na+i/K+i-mediated excitation-transcription coupling. Special attention was focused on the FOS gene and the possible mechanism of transcription regulation via G-quadruplexes, non-canonical secondary structures of nucleic acids, whose stability depends on [Na+]i/[K+]i-ratio. Verification of the [Na+]i/[K+]i-sensitive transcription regulation mechanism should be continued in forthcoming studies.

Ouabain is of cardiotonic steroids (CTS) family that is plant-derived compounds and is known for many years as therapeutic and cytotoxic agents. They are specific inhibitors of Na,K-ATPase, the enzyme, which pumps Na+ and K+ across plasma membrane of animal cells. Treatment of cells by CTS affects various cellular functions connected with the maintenance of the transmembrane gradient of Na+ and K+. Numerous studies demonstrated that binding of CTS to Na,K-ATPase not only suppresses its activity but also induces some signal pathways. This review is focused on different mechanisms of two ouabain effects: their ability (1) to protect rodent cells from apoptosis through the expression of [Na+]i-sensitive genes and (2) to trigger death of non-rodents cells (so-called «oncosis»), possessing combined markers of «classic» necrosis and «classic» apoptosis. Detailed study of oncosis demonstrated that the elevation of the [Na+]i/[K+]i ratio is not a sufficient for its triggering. Non-rodent cell death is determined by the characteristic property of “sensitive” to ouabain α1-subunit of Na,K-ATPase. In this case, ouabain binding leads to enzyme conformational changes triggering the activation of p38 mitogen-activated protein kinases (MAPK) signaling. The survival of rodent cells with ouabain-«resistant» α1-subunit is connected with another conformational transition induced by ouabain binding that results in the activation of ERK 1/2 signaling pathway.

Human adenovirus (HAdV) is a very common pathogen that typically causes minor disease in most patients. However, the virus can cause significant morbidity and mortality in certain populations, including young children, the elderly, and those with compromised immune systems. Currently, there are no approved therapeutics to treat HAdV infections, and the standard treatment relies on drugs approved to combat other viral infections. Such treatments often show inconsistent efficacy, and therefore, more effective antiviral therapies are necessary. In this review, we discuss recent developments in the search for new chemical and biological anti-HAdV therapeutics, including drugs that are currently undergoing preclinical/clinical testing, and small molecule screens for the identification of novel compounds that abrogate HAdV replication and disease.

Cardiotonic glycoside toxins, originally isolated from plants or amphibians, have been proposed as mammalian cardiotonic hormones. This paper is a review and update of the discovery of [i] a new class of steroid hormones, [ii] the path for their biosynthesis and [iii] some preliminary data on their function. The compounds are phosphoester conjugates and share a characteristic structural feature, a lactone ring, with [a] one class of synthetic potassium sparing diuretics and with [b] plant and amphibian cardiotonic steroids. Purification was initially monitored by cross reaction with steroid sulfate assays and later with digoxin-specific assays. Six compounds were purified by HPLC to near homogeneity and characterized by Tandem mass spectroscopy (MS-MS) and 31P-NMR. Three were digoxin-like materials (DLM) with 23 carbon atoms. The two extra carbon atoms form a spiral lactone E-ring. Several additional spiral lactones have been identified by MS-MS. In a pilot study, based on MS analysis, we evaluated phosphocholine steroid levels in individual serum samples in patients with pre-eclampsia (n=20). The control group of normotensive pregnant women (n=20) was used to estimate the mean and standard deviation. Twelve of the women with pre-eclampsia had a z-score over 2 for at least one of the four phosphocholine steroids. In contrast, only 1 sample from the normotensive women had a z-score over 2. The observation that there are two patterns, one with elevated phosphocholine steroid levels and one without the elevation, suggests that there may be two different underlying causes of pre-eclampsia. We now need to extend the study to determine which pattern predicts progression to life-threatening hypertension in pregnant women. This pilot study illustrates that it is possible to evaluate individual endogenous cardiotonic hormones without relying on antibodies developed to plant or amphibian toxins.

Hepatic fibrosis has been associated with a series of pathophysiological processes causing excessive accumulation of extracellular matrix proteins. Several cellular processes and molecular mechanisms have been implicated in the diseased liver that augments fibrogenesis, fibrogenic cytokines and associated liver complications. Liver biopsy remains an essential diagnostic tool for histological evaluation of hepatic fibrosis to establish a prognosis. In addition to being invasive, this methodology presents with several limitations including poor cost-effectiveness, prolonged hospitalizations, and risks of peritoneal bleeding, while the clinical use of this method does not reveal underlying pathogenic mechanisms. Several alternate noninvasive diagnostic strategies have been developed, to determine the extent of hepatic fibrosis, including the use of direct and indirect biomarkers. Immediate diagnosis of hepatic fibrosis by noninvasive means would be more palatable than a biopsy and could assist clinicians in taking early interventions timely, avoiding fatal complications, and improving prognosis. Therefore, we sought to review some common biomarkers of liver fibrosis along with some emerging candidates, including the oxidative stress-mediated biomarkers, epigenetic and genetic markers, exosomes, and miRNAs that needs further evaluation and would have better sensitivity and specificity. We also aim to elucidate the potential role of cardiotonic steroids (CTS) and evaluate the pro-inflammatory and profibrotic effects of CTS in exacerbating hepatic fibrosis. By understanding the underlying pathogenic processes, the efficacy of these biomarkers could allow for early diagnosis and treatment of hepatic fibrosis in chronic liver diseases, once validated.

Glioblastoma is a debilitating disease that is associated with poor prognosis and a very limited response to therapies; thus, molecularly targeted therapeutics and personalized therapy are urgently needed. The NaThe purpose of this narrative review is to explore the important roles of the sodium pump α1 subunit in glioblastoma and analyze its potential therapeutic applications.Expression of the sodium pump α1 subunit in glioblastoma tissues is generally higher than that in normal tissues. Sodium pump α1 subunit-mediated pivotal antiglioblastoma signaling pathways have been reviewed, and their impact on the sensitivity of glioblastoma cells to anticancer drugs has recently been clarified. In addition, various pharmacologically optimized sodium pump inhibitors have recently reached early clinical trials, and explorations of sodium pump α1 subunit inhibitors may hold promise for the development of stratification strategies in which patients are treated based on their isoform expression status.

Two prescient 1953 publications set the stage for the elucidation of a novel endocrine system: Schatzmann’s report that cardiotonic steroids (CTSs) are all Na+pump inhibitors, and Szent-Gyorgi’s suggestion that there is an endogenous “missing screw” in heart failure that CTSs like digoxin may replace. In 1977 I postulated that an endogenous Na+pump inhibitor acts as a natriuretic hormone and simultaneously elevates blood pressure (BP) in salt-dependent hypertension. This hypothesis was based on the idea that excess renal salt retention promoted the secretion of a CTS-like hormone that inhibits renal Na+pumps and salt reabsorption. The hormone also inhibits arterial Na+pumps, elevates myocyte Na+and promotes Na/Ca exchanger-mediated Ca2+gain. This enhances vasoconstriction and arterial tone—the hallmark of hypertension. Here I describe how those ideas led to the discovery that the CTS-like hormone is endogenous ouabain (EO), a key factor in the pathogenesis of hypertension and heart failure. Seminal observations that underlie the still-emerging picture of the EO-Na+pump endocrine system in the physiology and pathophysiology of multiple organ systems are summarized. Milestones include: 1) cloning the Na+pump isoforms and physiological studies of mutated pumps in mice; 2) discovery that Na+pumps are also EO-triggered signaling molecules; 3) demonstration that ouabain, but not digoxin, is hypertensinogenic; 4) elucidation of EO’s roles in kidney development and cardiovascular and renal physiology and pathophysiology; 5) discovery of “brain ouabain”, a component of a novel hypothalamic neuromodulatory pathway; and 6) finding that EO and its brain receptors modulate behavior and learning.

The group of compounds of the cardiotonic steroid (CTS) class includes steroid compounds that inhibit Na+,K+-ATPase. Recently, a large amount of data has been accumulating on the important role of CTS in regulating brain function through binding to Na+,K+-ATPase, which can perform a receptor role and trigger intracellular signaling cascades. Interestingly, it is in the brain where the greatest diversity of Na+,K+-ATPase isoforms different in their affinity for compounds of this class is observed. Despite the abundance of data on the presence of endogenous CTS in mammalian organisms and the evolutionary prerequisites for their existence, the presence of specific compounds is being actively discussed in the scientific literature, and enzymes directly involved in the synthesis of these compounds have not been identified. This review describes currently established functions of cardiotonic steroids and gives a detailed description of currently existing prerequisites of the endogenous origin of CTS in mammals. Experimental evidence of the presence of specific CTS in the body is given and attention is paid to the methods based on which the structure of endogenous CTS is established or predicted. We discuss why CTS can be important endogenous regulators in the nervous system and why the problem of isolating endogenous CTS from the brain and establishing their structure is urgent.

The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase α1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase α1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, α1, α2, and β1 protein content remained unchanged, and the cardiac Na/K-ATPase dose–response curve to ouabain shifted to the left as expected. In males aged 3–6 months, increased α1 sensitivity to CTS resulted in a significant increase in cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio accompanied by an increase in the myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the α1r/rα2s/s mouse failed to do so in the α1s/sα2s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase α1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions.

The endogenous Na+/K+-ATPase inhibitor, marinobufagenin (MBG), strongly associates with salt intake and a greater left ventricular mass index (LVMi) in humans and was shown to promote cardiac fibrosis and hypertrophy in animals. The adverse effects of MBG on cardiac remodeling may be exacerbated with obesity, due to an increased sensitivity of Na+/K+-ATPase to MBG. This study determined whether MBG is related to the change in LVMi over time in adults with a body mass index (BMI) &ge, 30 kg/m2 (obese) and &lt, 30 kg/m2 (non-obese). The study followed 275 healthy participants (aged 20&ndash, 30 years) from the African-Prospective study on the Early Detection and Identification of Cardiovascular disease and Hypertension (African-PREDICT) study over 4.5 years. At baseline, we measured 24 h urine MBG excretion. MBG levels were positively associated with salt intake. LVMi was determined by two-dimensional echocardiography at baseline and after &gt, 4.5 years. With multivariate adjusted analyses in obese adults (N = 56), we found a positive association of follow-up LVMi (Adjusted (Adj.) R2 = 0.35, Std. &beta, = 0.311, p = 0.007) and percentage change in LVMi (Adj. R2 = 0.40, = 0.336, p = 0.003) with baseline MBG excretion. No association of LVMi (Adj. R2 = 0.37, p = 0.85) or percentage change in LVMi (Adj. R2 = 0.19, p = 0.68) with MBG excretion was evident in normal weight adults (N = 123). These findings suggest that obese adults may be more sensitive to the adverse cardiac effects of MBG and provide new insight into the potential role of dietary salt, by way of MBG, in the pathogenesis of cardiac remodeling in obese individuals.

Melanoma is the most common cancer in young adults, with a constantly increasing incidence. Metastatic melanoma is a very aggressive cancer with a 5-year survival rate of about 22−25%. This is, in most cases, due to a lack of therapies which are effective on the long term. Hence, it is crucial to find new therapeutic agents to increase patient survival. Toad venoms are a rich source of potentially pharmaceutically active compounds and studies have highlighted their possible effect on cancer cells. We focused on the venoms of two different toad species: Bufo bufo and Rhinella marina. We screened the venom crude extracts, the fractions from crude extracts and isolated biomolecules by studying their antiproliferative properties on melanoma cells aiming to determine the compound or the combination of compounds with the highest antiproliferative effect. Our results indicated strong antiproliferative capacities of toad venoms on melanoma cells. We found that these effects were mainly due to bufadienolides that are cardiotonic steroids potentially acting on the Na+/K+ ATPase pump which is overexpressed in melanoma. Finally, our results indicated that bufalin alone was the most interesting compound among the isolated bufadienolides because it had the highest antiproliferative activity on melanoma cells., SCOPUS: ar.j, info:eu-repo/semantics/published

Ever since British Physician William Withering first described the use of foxglove extract for treatment of patients with congestive heart failure in 1785, cardiotonic steroids have been used clinically to treat heart failure and more recently atrial fibrillation. Due to their ability to bind and inhibit the ubiquitous transport enzyme sodium potassium pump, thus regulating intracellular Na+ concentration in every living cell, they are also an essential tool for research into the sodium potassium pump structure and function. Exogenous CTS have been clearly demonstrated to affect cardiovascular system through modulation of vagal tone, cardiac contraction (via ionic changes) and altered natriuresis. Reports of a number of endogenous CTS, since the 1980s, have intensified research into their physiologic and pathophysiologic roles and opened up novel therapeutic targets. Substantive evidence pointing to the role of endogenous ouabain and marinobufagenin, the two most prominent CTS, in development of cardiovascular disease has accumulated. Nevertheless, their presence, structure, biosynthesis pathways and even mechanism of action remain unclear or controversial. In this review the current state-of-the-art, the controversies and the remaining questions surrounding the role of endogenous cardiotonic steroids in health and disease are discussed.

High salt (HS) intake stimulates the production of marinobufagenin (MBG), an endogenous steroidal Na/K-ATPase ligand, which activates profibrotic signaling. HS is accompanied by a blood pressure (BP) increase in salt-sensitive hypertension, but not in normotensive animals. Here, we investigated whether HS stimulates MBG production and activates transforming growth factor-beta (TGF-&beta, ) profibrotic signaling in young normotensive rats, and whether these changes can be reversed by reducing salt to a normal salt (NS) level. Three-month old male Sprague&ndash, Dawley rats received NS for 4 and 8 weeks (0.5% NaCl, NS4 and NS8), or HS for 4 and 8 weeks (4% NaCl, HS4 and HS8), or HS for 4 weeks followed by NS for 4 weeks (HS4/NS4), n = 8/group. Systolic BP (SBP), pulse wave velocity (PWV), MBG excretion, aortic collagen 1&alpha, 2, collagen 4&alpha, 1 and TGF-&beta, Smad2, Smad3, Fli-1 mRNA, and total collagen abundance were measured at baseline (BL), and on weeks 4 and 8. Statistical analysis was performed using one-way ANOVA. SBP was not affected by HS (125 &plusmn, 5 and 126 &plusmn, 6 vs. 128 &plusmn, 7 mmHg, HS4 and HS8 vs. BL, p &gt, 0.05). HS increased MBG (164 &plusmn, 19 vs. 103 &plusmn, 19 pmol/24 h/kg, HS4 vs. BL, p &lt, 0.05) and PWV (3.7 &plusmn, 0.2 vs. 2.7 &plusmn, 0.2 m/s, HS4 vs. NS4, p &lt, 0.05). HS8 was associated with a further increase in MBG and PWV, with an increase in aortic Col1a2 80%), Col4a1 (50%), Tgfb1 (30%), Smad2 (30%) and Smad3 (45%) mRNAs, and aortic wall collagen (180%) vs. NS8 (all p &lt, 0.05). NS following HS downregulated HS-induced factors: in HS4/NS4, the MBG level was 91 &plusmn, 12 pmol/24 h/kg (twofold lower than HS8, p &lt, 0.01), PWV was 3.7 &plusmn, 0.3 vs. 4.7 &plusmn, 0.2 m/s (HS4/NS4 vs. HS8, p &lt, 0.05), aortic wall Tgfb1, Col1a2, Col4a1, Smad2, Smad3 mRNAs, and collagen abundance were reversed by salt reduction to the BL levels (p &lt, 0.05). HS was associated with an activation of TGF-&beta, signaling, aortic fibrosis and aortic stiffness accompanied by an MBG increase in the absence of SBP changes in young normotensive rats. The reduction of dietary salt following HS decreased MBG, PWV, aortic wall collagen and TGF-&beta, Thus, HS-induced aortic stiffness in normotensive animals occurred in the context of elevated MBG, which may activate SMAD-dependent TGF-&beta, pro-fibrotic signaling. This data suggests that a decrease in salt consumption could help to restore aortic elasticity and diminish the risk of cardiovascular disease by reducing the production of the pro-fibrotic factor MBG.

Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na+/K+-ATPase inhibition, they activate signal transduction via protein&ndash, protein interactions. Diversity of endogenous bufadienolides and mechanisms of action may indicate the presence of functional selectivity and unique cellular outcomes. We evaluated whether the bufadienolides telocinobufagin and marinobufagin induce changes in proliferation or viability of pig kidney (LLC-PK1) cells and the mechanisms involved in these changes. In some experiments, ouabain was used as a positive control. CTS exhibited an inhibitory IC50 of 0.20 (telocinobufagin), 0.14 (ouabain), and 3.40 &mu, M (marinobufagin) for pig kidney Na+/K+-ATPase activity and concentrations that barely inhibited it were tested in LLC-PK1 cells. CTS induced rapid ERK1/2 phosphorylation, but corresponding proliferative response was observed for marinobufagin and ouabain instead of telocinobufagin. Telocinobufagin increased Bax:Bcl-2 expression ratio, sub-G0 cell cycle phase and pyknotic nuclei, indicating apoptosis. Src and MEK1/2 inhibitors blunted marinobufagin but not telocinobufagin effect, which was also not mediated by p38, JNK1/2, and PI3K. However, BIO, a GSK-3&beta, inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3&beta, at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/&beta, catenin pathway by acting upstream to &beta, catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity.

Background: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. Objectives and Methods: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 &plusmn, 4 mmHg, age 29 &plusmn, 2 years, 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 &plusmn, 2 mmHg, controls) were enrolled in the clinical study. Results: PE was associated with a higher placental (0.04 &plusmn, 0.01 vs. 0.49 &plusmn, 0.11 pmol/g, p &lt, 0.01) and plasma MBG (0.5 &plusmn, 0.1 vs. 1.6 &plusmn, 0.5 nmol/L, 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 &plusmn, 0.2 vs. 1.5 &plusmn, 0.2 &micro, mol Pi/mL/hr, 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p &lt, 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p &lt, 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. Conclusion: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.

Cardiotonic steroids (CTS) are Na+/K+-ATPase (NKA) ligands that are elevated in volume-expanded states and associated with cardiac and renal dysfunction in both clinical and experimental settings. We test the hypothesis that the CTS telocinobufagin (TCB) promotes renal dysfunction in a process involving signaling through the NKA &alpha, 1 in the following studies. First, we infuse TCB (4 weeks at 0.1 &micro, g/g/day) or a vehicle into mice expressing wild-type (WT) NKA &alpha, 1, as well as mice with a genetic reduction (~40%) of NKA &alpha, 1 (NKA &alpha, 1+/&minus, ). Continuous TCB infusion results in increased proteinuria and cystatin C in WT mice which are significantly attenuated in NKA &alpha, mice (all p &lt, 0.05), despite similar increases in blood pressure. In a series of in vitro experiments, 24-h treatment of HK2 renal proximal tubular cells with TCB results in significant dose-dependent increases in both Collagens 1 and 3 mRNA (2-fold increases at 10 nM, 5-fold increases at 100 nM, p &lt, 0.05). Similar effects are seen in primary human renal mesangial cells. TCB treatment (100 nM) of SYF fibroblasts reconstituted with cSrc results in a 1.5-fold increase in Collagens 1 and 3 mRNA (p &lt, 0.05), as well as increases in both Transforming Growth factor beta (TGFb, 1.5 fold, p &lt, 0.05) and Connective Tissue Growth Factor (CTGF, 2 fold, p &lt, 0.05), while these effects are absent in SYF cells without Src kinase. In a patient study of subjects with chronic kidney disease, TCB is elevated compared to healthy volunteers. These studies suggest that the pro-fibrotic effects of TCB in the kidney are mediated though the NKA-Src kinase signaling pathway and may have relevance to volume-overloaded conditions, such as chronic kidney disease where TCB is elevated.

It is likely that the processes of steroid behavior are influenced differently by a particular steroid structural modification. This chapter reviews the contribution of theoretical investigations toward the understanding of at least a part of the interrelations and toward the explanation of at least a part of the existing biological data. Basic and more sophisticated approaches have been developed and applied to establish quantitative relationships between chemical structure and observed biological activity data. For a series of compounds that vary only in the substituents at one or more substitution sites, it is an obvious aim to assign to each substituent an individual contribution to the biological activity. Pattern recognition methods as well as linear and quadratic discriminant analyses were used by D. R. Henry and J. H. Block to classify 46 compounds as estrogens, androgens, progestins, corticoids, or cardiotonic steroids. All the structure and potency data of unusually configurated steroids can be expected to permit improvement in future modeling attempts.

Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner.

Toads are among several groups of organisms chemically defended with lethal concentrations of cardiotonic steroids. As a result, most predators that prey on amphibians avoid toads. However, several species of snakes have gained resistance-conferring mutations of Na+/K+-ATPase, the molecular target of cardiotonic steroids, and can feed on toads readily. Despite recent advances in our understanding of this adaptation at the genetic level, we have lacked functional evidence for how mutations of Na+/K+-ATPase account for cardiotonic steroid resistance in snake tissues. To address this issue, it is necessary to determine how the Na+/K+-ATPases of snakes react to the toxins. Some tissues might have Na+/K+-ATPases that are more susceptible than others and can thus provide clues about how the toxins influence organismal function. Here we provide a mechanistic link between observed Na+/K+-ATPase substitutions and observed resistance using actual snake Na+/K+-ATPases. We used an in vitro approach to determine the tissue-specific levels of sensitivity to cardiotonic steroids in select resistant and non-resistant snakes. We compared the sensitivities of select tissues within and between species. Our results suggest that resistant snakes contain highly resistant Na+/K+-ATPases in their heart and kidney, both of which rely heavily on the enzymes to function, whereas tissues that do not rely as heavily on Na+/K+-ATPases or might be protected from cardiotonic steroids by other means (liver, gut, and brain) contain non-resistant forms of the enzyme. This study reveals functional evidence that tissue-level target-site insensitivity to cardiotonic steroids varies not only among species but also across tissues within resistant taxa.

The Na/K-ATPase (NKA), or Na pump, is a member of the P-type ATPase superfamily. In addition to pumping ions across cell membrane, it is engaged in assembly of multiple protein complexes in the plasma membrane. This assembly allows NKA to perform many non-pumping functions including signal transduction that are important for animal physiology and disease progression. This article will focus on the role of protein interaction in NKA-mediated signal transduction, and its potential utility as target for developing new therapeutics.

Lung cancer is a leading cause of global cancer deaths. Na/K-ATPase has been studied as a target for cancer treatment. Cardiotonic steroids (CS) trigger intracellular signalling upon binding to Na/K-ATPase. Normal lung and tumour cells frequently express different pump isoforms. Thus, Na/K-ATPase is a powerful target for lung cancer treatment. Drugs targeting Na/K-ATPase may induce apoptosis and autophagy in transformed cells. We argue that Na/K-ATPase has a role as a potential target in chemotherapy in lung cancer treatment. We discuss the effects of Na/K-ATPase ligands and molecular pathways inducing deleterious effects on lung cancer cells, especially those leading to apoptosis and autophagy.

Other than genetic regulation of salt sensitivity of blood pressure, many factors have been shown to regulate renal sodium handling which contributes to long-term blood pressure regulation and have been extensively reviewed. Here we present our progress on the Na/K-ATPase signaling mediated sodium reabsorption in renal proximal tubules, from cardiotonic steroids-mediated to reactive oxygen species (ROS)-mediated Na/K-ATPase signaling that contributes to experimental salt sensitivity.

A synthetic tripodal-based thiourea receptor (PNTTU) was used to explore the receptor/ligand binding affinity using CTB cells. The human extravillous CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. The cell proliferation, migration and angiogenic factors were evaluated in PNTTU-treated CTB cells. The PNTTU inhibited the CTBs proliferation and migration. The soluble fms-like tyrosine kinase-1 (sFlt-1) secretion was increased while vascular endothelial growth factor (VEGF) was decreased in the culture media of CTB cells treated with ≥1 nM PNTTU. The angiotensin II receptor type 2 (AT2) expression was significantly upregulated in ≥1 nM PNTTU-treated CTB cells in compared to basal, however, the angiotensin II receptor, type 1 (AT1) and vascular endothelial growth factor receptor 1 (VEGFR-1) expression was downregulated. The anti-proliferative and anti-angiogenic effect of this compound on CTB cells are similar to the effect of CTSs. The receptor/ligand affinity of PNTTU on CTBs provides us the clue to design a potent inhibitor to prevent the CTS-induced impairment of CTB cells.

Cardiac glycosides, also known as cardiotonic steroids, are a group of natural products that share a steroid-like structure with an unsaturated lactone ring and the ability to induce cardiotonic effects mediated by a selective inhibition of the Na+/K+-ATPase. Cardiac glycosides have been used for many years in the treatment of cardiac congestion and some types of cardiac arrhythmias. Recent data suggest that cardiac glycosides may also be useful in the treatment of cancer. These compounds typically inhibit cancer cell proliferation at nanomolar concentrations, and recent high-throughput screenings of drug libraries have therefore identified cardiac glycosides as potent inhibitors of cancer cell growth. Cardiac glycosides can also block tumor growth in rodent models, which further supports the idea that they have potential for cancer therapy. Evidence also suggests, however, that cardiac glycosides may not inhibit cancer cell proliferation selectively and the potent inhibition of tumor growth induced by cardiac glycosides in mice xenografted with human cancer cells is probably an experimental artifact caused by their ability to selectively kill human cells versus rodent cells. This paper reviews such evidence and discusses experimental approaches that could be used to reveal the cancer therapeutic potential of cardiac glycosides in preclinical studies.

The Na/K-ATPase as an essential ion pump was discovered more than 50 years ago (Skou (1989) Biochim. Biophys. Acta 1000, 439-446; Feraille and Doucet (2001) Physiol. Rev. 81, 345-418). The signaling function of Na/K-ATPase has been gradually appreciated over the last 20 years, first from the studies of regulatory effects of ouabain on cardiac cell growth. Several reviews on this topic have been written during the last few years (Schoner and Scheiner-Bobis (2007) Am. J. Physiol. Cell. Physiol. 293, C509-C536; Xie and Cai (2003) Mol. Interv. 3, 157 - 168; Bagrov et al. (2009) Pharmacol. Rev. 61, 9-38; Tian and Xie (2008) Physiology 23, 205-211; Fontana et al. (2013) FEBS J. 280, 5450-5455; Blanco and Wallace (2013) Am. J. Physiol. Renal Physiol. 305, F797-F812). This article will focus on the molecular mechanism of Na/K-ATPase-mediated signal transduction and its potential regulatory role in renal physiology and diseases.

Objective— Circulating levels of cardiotonic steroids (CTS) are elevated in various chronic inflammatory conditions, but the role of CTS in inflammation remains largely unknown. We have previously shown that the CTS ouabain stimulates proinflammatory responses in murine macrophages. In this study, we aim to explore the mechanism how CTS induce proinflammatory responses in primary murine and human macrophages. Approach and Results— Using both murine peritoneal macrophages and human monocyte–derived macrophages, we demonstrated that ouabain activated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), leading to proinflammatory cytokine (eg, MCP-1 [monocyte chemotactic protein 1], TNF-α [tumor necrosis factor-α], IL-1β [interleukin-1β], and IL-6) production. By applying siRNA techniques and murine peritoneal macrophages isolated from genetically modified mice, we showed that macrophages partially deficient in Na/K-ATPase, the receptor for CTS, or fully deficient in the scavenger receptor CD36 or TLR4 (Toll-like receptor) were resistant to ouabain-induced NF-κB activation, suggesting an indispensable role of these 3 receptors in this pathway. Mechanistically, this effect of ouabain was independent of the ion transport function of the Na/K-ATPase. Instead, ouabain stimulated a signaling complex, including Na/K-ATPase, CD36, and TLR4. Subsequently, TLR4 recruited MyD88 adaptor protein for NF-κB activation. Furthermore, intraperitoneal injection of ouabain into mice specifically recruited Ly6C + CCR2 + monocyte subtypes to the peritoneal cavities, indicating that the CTS ouabain triggers inflammation in vivo. Conclusions— CTS activate NF-κB leading to proinflammatory cytokine production in primary macrophages through a signaling complex, including CD36, TLR4, and Na/K-ATPase. These findings warrant further studies on endogenous CTS in chronic inflammatory diseases, such as atherosclerosis.

Cardiotonic steroids (CTS) are a class of specific ligands of the Na(+), K(+)- ATPase (NKA). NKA is a P-type ATPase that is ubiquitously expressed and although well known to be responsible for the maintenance of the cell electrochemical gradient through active transport, NKA can also act as a signal transducer in the presence of CTS. Inflammation, in addition to importantly driving organism defense and survival mechanisms, can also modulate NKA activity and memory formation, as well as being relevant to many chronic illnesses, neurodegenerative diseases, and mood disorders. The aim of the current review is to highlight the recent advances as to the role of CTS and NKA in inflammatory process, with a particular focus in the central nervous system.

It has been known for some time that dietary salt intake correlates with the prevalence of cardiovascular disease. However, the molecular link between dietary salt and cardiovascular disease is poorly understood. On this background, it has been observed that there are a class of hormones called cardiotonic steroids whose concentrations increase in response to increases in dietary salt. We have shown that some of these hormones may be natriuretic, but we have also shown that they may also be responsible for progressive renal and cardiac injury. Based on data summarized in this review, we propose cardiotonic steroids may serve as the molecular link between dietary salt and cardiovascular disease.

High dietary salt intake has been listed among the top ten risk factors for disability-adjusted life years. We discuss the role of endogenous cardiotonic steroids in mediating the dietary salt-induced hypertension and organ damage.

The main properties of Na+/K+-ATPase as a natural receptor for cardiotonic steroids are considered. Primary attention is focused on structural and functional differences between the α-subunit isoforms of Na+/K+-ATPase in different tissues. General information on the role of the Na+ pump in signaling cascades in kidney epithelial cells, cardiomyocytes and neurons is presented. The data obtained indicate that, in neurons, several α-isoforms of Na+/K+-ATPase possessing different sensitivity to ouabain may have different signaling functions.

In 1972 Neal Bricker presented the “trade-off” hypothesis in which he detailed the role of physiological adaptation processes in mediating some of the pathophysiology associated with declines in renal function. In the late 1990’s Xie and Askari published seminal studies indicating that the Na+/K+-ATPase (NKA) was not only an ion pump, but also a signal transducer that interacts with several signaling partners. Since this discovery, numerous studies from multiple laboratories have shown that the NKA is a central player in mediating some of these long-term “trade-offs” of the physiological adaptation processes which Bricker originally proposed in the 1970’s. In fact, NKA ligands such as cardiotonic steroids (CTS), have been shown to signal through NKA, and consequently been implicated in mediating both adaptive and maladaptive responses to volume overload such as fibrosis and oxidative stress. In this review we will emphasize the role the NKA plays in this “trade-off” with respect to CTS signaling and its implication in inflammation and fibrosis in target organs including the heart, kidney, and vasculature. As inflammation and fibrosis exhibit key roles in the pathogenesis of a number of clinical disorders such as chronic kidney disease, heart failure, atherosclerosis, obesity, preeclampsia, and aging, this review will also highlight the role of newly discovered NKA signaling partners in mediating some of these conditions.

Current salt intake is too high. Current evidence documents that salt is crucial to the genesis of hypertension.

(1) Background: Cardiotonic steroids have been found to stimulate collagen synthesis and might be potential wound healing therapeutics. The objective of this study was to evaluate the feasibility of digitoxigenin and its topical formulation for wound healing; (2) Methods: In the in vitro study, the human dermal fibroblast cells were treated with digitoxigenin and collagen synthesis was assessed. In the in vivo study, digitoxigenin was applied to excisional full-thickness wounds in rats immediately after wounding and remained for three days, and wound open was evaluated over 10 days. A digitoxigenin formulation for topical administration was prepared, and the in vitro release and in vivo wound healing effect were investigated; (3) Results: The expression of procollagen in human dermal fibroblast was significantly increased with the exposure to 0.1 nM digitoxigenin. Topical application of digitoxigenin in olive oil or alginate solution for three days significantly decreased the wound open in rats. Similarly, topical administration of the developed digitoxigenin formulation for three days also significantly increased wound healing. No wound healing effects were observed at days 7 and 10 after wounding when digitoxigenin was not applied; and, (4) Conclusions: It was possible to deliver digitoxigenin using the developed formulation. However, the wound healing effect of digitoxigenin and its mechanisms need to be further investigated in future studies.

Preeclampsia is a major cause of maternal and fetal mortality, and its pathogenesis is not fully understood. Endogenous digitalis-like cardiotonic steroids (CTS) have been implicated in the pathophysiology of preeclampsia; this is illustrated by clinical observations that Digibind, a therapeutic digoxin antibody fragment which binds CTS, lowers blood pressure and reverses Na/K-ATPase inhibition in patients with preeclampsia. Recently we reported that plasma levels of marinobufagenin (MBG), a bufadienolide vasoconstrictor CTS, are increased four-fold in patients with severe preeclampsia.In the present study, we compared levels of MBG in normal and preeclamptic placentae, as well as the interactions of Digibind and antibodies against MBG and ouabain with material purified from preeclamptic placentae using high-performance liquid chromatography (HPLC).Levels of endogenous MBG, but not that of endogenous ouabain, exhibited a four-fold elevation in preeclamptic placentae vs. normal placentae (13.6 +/- 2.5 and 48.6 +/- 7.0 nmoles/g tissue; P0.01). The elution time of endogenous placental MBG-like immunoreactive material from reverse-phase HPLC column was identical to that of authentic MBG. A competitive immunoassay based on Digibind exhibited reactivity to HPLC fractions having retention times similar to that seen with MBG and other bufadienolides, but not to ouabain-like immunoreactive material.Our results suggest that elevated levels of endogenous bufadienolide CTS represent a potential target for immunoneutralization in patients with preeclampsia.

We previously reported that cardiotonic steroids stimulate collagen synthesis by cardiac fibroblasts in a process that involves signaling through the Na-K-ATPase pathway (Elkareh et al. Hypertension 49: 215–224, 2007). In this study, we examined the effect of cardiotonic steroids on dermal fibroblasts collagen synthesis and on wound healing. Increased collagen expression by human dermal fibroblasts was noted in response to the cardiotonic steroid marinobufagenin in a dose- and time-dependent fashion. An eightfold increase in collagen synthesis was noted when cells were exposed to 10 nM marinobufagenin for 24 h ( P < 0.01). Similar increases in proline incorporation were seen following treatment with digoxin, ouabain, and marinobufagenin (10 nM × 24 h, all results P < 0.01 vs. control). The coadministration of the Src inhibitor PP2 or N-acetylcysteine completely prevented collagen stimulation by marinobufagenin. Next, we examined the effect of digoxin, ouabain, and marinobufagenin on the rate of wound closure in an in vitro model where human dermal fibroblasts cultures were wounded with a pipette tip and monitored by digital microscopy. Finally, we administered digoxin in an in vivo wound healing model. Olive oil was chosen as the digoxin carrier because of a favorable partition coefficient observed for labeled digoxin with saline. This application significantly accelerated in vivo wound healing in rats wounded with an 8-mm biopsy cut. Increased collagen accumulation was noted 9 days after wounding (both P < 0.01). The data suggest that cardiotonic steroids induce increases in collagen synthesis by dermal fibroblasts, as could potentially be exploited to accelerate wound healing.

Several endogenous digitalis-like cardiotonic steroids (CTS) have been identified in humans, including endogenous ouabain, a cardenolide, and marinobufagenin which belongs to a class of bufadienolides. The main function of CTS is maintenance of sodium homeostasis and regulation of natriuretic function. Endogenous cardiotonic steroids bind to the Na/K-ATPase and exhibit two kinds of effects, inhibition of transmembrane transport of monovalent cations, and induction of a complex cascade of cell signaling. In Dahl salt-sensitive rats with NaCl-induced hypertension, endogenous ouabain, acting as a neurohormone stimulates adrenocortical marinobufagenin, which raises blood pressure via inhibition of vascular Na/K-ATPase. In a subgroup of hypertensive subjects, which possess a polymorphism of alpha-adducin gene, elevated levels of endogenous ouabain activate renotubular Na/K-ATPase and induce renal sodium retention. Elevated levels of CTS also contribute to pathogenesis of congestive heart failure, preeclampsia, and diabetes mellitus. The approaches to pharmacological antagonism of the effects of CTS include blockade of CTS receptor site on the Na/K-ATPase, immunoneutralization, and interactions at the levels of Na/K-ATPase phosphorylation/dephosphorylation by protein kinases С and G.

Cardiotonic steroids (CS) such as ouabain, digoxin and bufalin, are steroidal drugs prepared from the seeds and dried leaves of the genus Digitalis, and the skin and parotid gland of amphibians, are used as a cardiac stimulant. Steroids similar or identical to the cardiotonic steroids were identified in human tissues. The available literature unequivocally supports the notion that these endogenous CS function as hormones in mammals. Recent studies show that although similar in structure, the different CS exhibit diverse biological responses. This was shown at the molecular, cellular, tissue and whole animal levels. This review summarizes these diversities, raises a possible explanation for their presence and discusses their implication on the physiological role of the different steroids.

The sodium pump, Na(+)/K(+)-ATPase, could be an important target for the development of anti-cancer drugs as it serves as a versatile signal transducer, it is a key player in cell adhesion and its aberrant expression and activity are implicated in the development and progression of different cancers. Cardiotonic steroids, known ligands of the sodium pump have been widely used for the treatment of heart failure. However, early epidemiological evaluations and subsequent demonstration of anti-cancer activity in vitro and in vivo have indicated the possibility of developing this class of compound as chemotherapeutic agents in oncology. Their development to date as anti-cancer agents has however been impaired by a narrow therapeutic margin resulting from their potential to induce cardiovascular side-effects. The review will thus discuss (i) sodium pump structure, function, expression in diverse cancers and its chemical targeting and that of its sub-units, (ii) reported in vitro and in vivo anti-cancer activity of cardiotonic steroids, (iii) managing the toxicity of these compounds and the limitations of existing preclinical models to adequately predict the cardiotoxic potential of new molecules in man and (iv) the potential of chemical modification to reduce the cardiovascular side-effects and improve the anti-cancer activity of new molecules.

The Dahl salt-sensitive rat is a well-established model to study essential hypertension. We first described a subgroup of these rats based on the unique response pattern in systolic blood pressure during the first weeks of exposure to a high salt diet that included cataract formation. We classified this group as cataract-prone Dahl salt-sensitive rat. We also were able to predict and prevent cataract formation in these rats. Further studies showed an inhibition of lens Na, K-ATPase activity which may be in part responsible for the cataract formation. Other studies in Dahl salt-sensitive rats maintained on a high salt diet have also shown decreased Na, K-ATPase activity in several tissues and increased levels of endogenous circulating Na, K pump inhibitors. For over 20 years, endogenous cardiotonic steroids have been postulated to inhibit Na, K-ATPase in both humans as well as in experimental animal models of hypertension. Recent findings have shown results suggesting that there are several forms of cardiotonic steroids with minor differences in structural functionalities, site of production, and specific pump selectivity. We present original data that supports a role for cardiotonic steroids in disease progression related to increased salt-sensitivity. We found increased levels of free endogenous cardiotonic steroids in those rats that were classified as cataract-prone according to their initial systolic blood pressure response to a high salt intake when compared to non-cataract prone Dahl salt-sensitive rats and their control Dahl salt-resistant rats. The cataract-prone Dahl salt-sensitive rat is an animal model that can help and contribute to open a new door to possibly elucidate the role of endogenous cardiotonic steroids in the pathogenesis and progression of diseases related to salt-sensitive hypertension.

Recently, cardiotonic steroids (CTS), used in heart failure treatment, have shown interesting antitumor effect acting by several intracellular signaling pathways triggered by Na, K-ATPase. Several signaling pathways activated by CTS are involved in tumor progression, metastasis, and proliferation, leading an interesting perspective to study the relationship of the Na, K-ATPase and cancer. Here we highlight the major signaling pathways modulated by Na, K-ATPase and the chemical modification of traditional CTS that are under study to develop new anticancer drugs.

The link between renal sodium excretion and body fluid volumes has been a topic of interest since the early days of renal physiology (1). Glomerular filtration rate (GFR) and aldosterone were the first two controllers of renal sodium excretion to be recognized. A “third factor” was discovered when de Wardener and colleagues showed that volume expansion natriuresis still occurred in dogs given supramaximal doses of mineralocorticoids and without an increase in GFR (2). de Wardener et al. suggested that the natriuresis was due to a “natriuretic hormone” (NH), launching a new field of investigation. NH was thus defined as a compound that circulates in blood, the level of which is regulated appropriately by changes in sodium and water balance. The nine chapters that follow highlight several areas in which the NH field has subsequently developed. First, de Wardener’s original experiments were refined to control potential factors other than GFR and aldosterone. These studies, reviewed by Lichardus (3), confirmed the original observation, and also led to the discovery of the so-called “physical factors” that affect sodium excretion (4). Second, attempts to purify NH from various tissues and body fluids, as summarized in Table 1 by Hamlyn (5), led to the discovery of two important families of factors, atrial natriuretic peptides (ANP), and endogenous cardiotonic steroids (CTS). The discovery of ANP led rapidly to its characterization as a family of peptides with three major components: ANP derived from cardiac tissue, BNP from brain, and CNP from endothelium (6). This complex system of natriuretic vasodilators, discussed in the chapters by Hamlyn (5), and Hodes and Lichstein (6), continues to be of interest as an NH, a neurotransmitter (6), and a factor with many other functions (7). In sharp contrast, the identification of endogenous CTS as circulating Na, K ATPase inhibitors has been the source of considerable controversy (8). Two structural classes of CTS have been identified: authentic ouabain, and the bufodienolides, originally identified in toads (9). As discussed by Hamlyn, a bufodienolide is the more likely candidate NH, whereas ouabain in physiologic concentration appears to be antinatriuretic (5). CTS cause vasoconstriction by a mechanism proposed by Blaustein et al. (10) and endogenous CTS have been implicated in the pathophysiology of hypertension (9). Despite overwhelming evidence to the contrary, however, difficulty identifying ouabain in biological fluids continues to be reported (11, 12). At least three other natriuretic compounds related to control of sodium balance have been identified. As discussed by Gonick (13), a natriuretic protein associated with various forms of human hypertension consists of a 408 Da compound and a 12 kDa carrier protein. The identity of these compounds has not been completed (see below). Two xanthurenic acid derivatives (MW 368 and 284), discussed by Bricker et al. (14), are potential regulators of sodium balance in chronic renal failure. Dietary sodium releases two natriuretic factors from the gastrointestinal tract identified as guanylin and uroguanylin (MW 10.3 and 1.7 kDa), discussed in the paper by Hodes and Lichstein (6). Two other types of natriuretic compounds that inhibit Na, K ATPase, but are not natriuretic hormones as defined above, are two vanadium diascorbates (MW approximately 400), and two spherical oligo silicic acids (SOSA) (MW 408). These interesting compounds of uncertain significance are described in papers by their discoverers Kramer (15) and Kerek and Voicu (16), respectively. The apparent identical molecular weight of SOSA and the compound isolated by Gonick is possibly a coincidence. Alternatively, it may be related to the trace amounts of silicon in human plasma (17) and have significance beyond that implied by the serendipitous discovery of SOSA. A third area is studies of the physiological mechanism(s) by which these factors might function as natriuretic hormones. ANP release is controlled by stretch of the cardiac atria (18), and in that regard is a classic, volume controlled, NH system as originally conceived (19). Although evidence indicates that CTS are released in response to increased sodium intake (5), no causal mechanism connecting sodium intake and CTS release has been demonstrated. As noted by Hamlyn (5), cerebrospinal fluid sodium concentration controls the release of an unidentified natriuretic factor from brain, causing “CNS natriuresis.” The possible role of CTS or some other NH in this phenomenon should be explored. A fourth area of investigation is studies of collateral, pathophysiological, effects of the various factors. Both ANP and CTS are vasoactive and have been implicated in the pathophysiology of hypertension (5, 6, 9). Since both appear to be neurotransmitters or neuromodulators (6), their role in hypertension may have both central and peripheral components. Pathological effects of excess CTS in the CNS may also include an etiologic role in mood disorders, including depression and bipolar disorder (6). Finally, the signaling effects of CTS on Na, K ATPase not dependent on ion pumping, mediated by activation of the tyrosine kinase Src and other signaling molecules, is discussed by Xie et al. (20). This pathway is involved in the natriuretic effect of bufodienolides (21), and is implicated in pathophysiological processes, such as oxidative stress, and organ fibrosis (20). de Wardener’s original hypothesis has led to the discovery of a rich array of factors with multiple biologic activities. ANP is the only classical NH described so far. Future work, including the development of methods for antagonizing these factors, should increase our understanding of the regulation of renal sodium excretion and blood pressure and offer novel treatments for several clinical disorders.

Foxglove and its constituents therapeutic agent digitalis have been used for centuries for the treatment of heart failure. All digitalis-like cardiotonic steroids enhance heart contraction through a mechanism involving the inhibition of the Na(+),K(+)- ATPase. Recently, Rathore and colleagues reported that sex-based differences may exist in the efficacy of digoxin for the treatment of heart failure. The authors of the study found that female patients exhibited increased risk of death associated with digoxin therapy, whereas male patients appeared to have no increased risk of death related to digoxin therapy. Blaustein and colleagues delve into the report and discuss possible explanations for these findings, suggest alternative ones, and advocate for enrolling greater numbers of women in clinical studies.

The cytotrophoblast (CTB) cells of the human placenta have membrane receptors that bind certain cardiotonic steroids (CTS) found in blood plasma. One of these, marinobufagenin, is a key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR) to study their effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous CTB cells (Sw.71) used in this study were derived from first trimester chorionic villus tissue. Culture media of CTB cells treated with ≥1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1) was significantly increased while VEGF (vascular endothelial growth factor) was significantly decreased in the culture media (* p < 0.05 for each) The AT2 receptor (Angiotensin II receptor type 2) expression was significantly upregulated in ≥1 nM SR-treated CTB cells as compared to basal; however, the AT1 (Angiotensin II receptor, type 1) and VEGFR-1 (vascular endothelial growth factor receptor 1) receptor expression was significantly downregulated (* p < 0.05 for each). Our results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced impairment of CTB cells.

It has been postulated for some time that endogenous digitalis-like substances, also called cardiotonic steroids (CTS), exist, and that these substances are involved in sodium handling. Within the past 20 years, these substances have been unequivocally identified and measurements of circulating and tissue concentrations have been made. More recently, it has been identified that CTS also mediate signal transduction through the Na/K-ATPase, and consequently been implicated in profibrotic pathways. This review will discuss the mechanism of CTS in renal sodium handling and a potential "trade-off" effect from their role in inducing tissue fibrosis.

Objective: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy with abnormal placentation. We have demonstrated that cardiotonic steroids (CTSs) impair cytotrophoblast (CTB) function...

A short review on cardiotonic steroids and their analogues is presented. The natural, semisynthetic and synthetic derivatives, as well as their mechanism of action and structure-activity relationships are shown, with a special reference to aminoguanidine derivatives.

Gender difference has been suggested as a risk factor for developing cardiovascular and renal diseases in humans and experimental animals. As a major sex hormone, progesterone was reported to compete with cardiotonic steroid binding to Na/K-ATPase. Our previous publication demonstrated that cardiotonic steroids (e.g., marinobufagenin) play an important role in the development of experimental uremic cardiomyopathy. We also observed that the putative mineralocorticoid antagonists, spironolactone and its major metabolite canrenone, antagonize binding of cardiotonic steroids to Na/K-ATPase in a competitive manner and also ameliorate experimental uremic cardiomyopathy induced by partial nephrectomy. In the following studies, we noted that progesterone displayed competitive inhibition of cardiotonic steroid binding to Na/K-ATPase and partially inhibited collagen synthesis induced by marinobufagenin in cultured cardiac fibroblasts. Therefore, we sought to examine whether female rats displayed less uremic cardiomyopathy than male rats when subjected to partial nephrectomy. Although partial nephrectomy caused the induction of smaller increases in blood pressure of female rats, they appeared to be similarly susceptible to cardiac remodeling induced by partial nephrectomy in terms of hypertrophy and fibrosis as age-matched male rats. The possible explanations for our findings are therefore discussed.