Oleandrin suppresses activation of nuclear transcription factor-kappaB, activator protein-1, and c-Jun NH2-terminal kinase.
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Agents that can suppress the activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) may be able to block tumorigenesis and inflammation. Oleandrin, a polyphenolic cardiac glycoside derived from the leaves of Nerium oleander, is a candidate NF-kappaB and AP-1 modulator. We investigated the effect of oleandrin on NF-kappaB activation induced by inflammatory agents. Oleandrin blocked tumor necrosis factor (TNF)-induced activation of NF-kappaB in a concentration- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of IkappaBalpha, an inhibitor of NF-kappaB. A proprietary hot water extract of oleander (Anvirzel) also blocked TNF-induced NF-kappaB activation; subsequent fractionation of the extract revealed that this activity was attributable to oleandrin. The effects of oleandrin were not cell type specific, because it blocked TNF-induced NF-kappaB activation in a variety of cells. NF-kappaB-dependent reporter gene transcription activated by TNF was also suppressed by oleandrin. The TNF-induced NF-kappaB activation cascade involving TNF receptor 1/TNF receptor-associated death domain/TNF receptor-associated factor 2/NF-kappaB-inducing kinase/IkappaBalpha kinase was interrupted at the TNF receptor-associated factor 2 and NF-kappaB-inducing kinase sites by oleandrin, thus suppressing NF-kappaB reporter gene expression. Oleandrin blocked NF-kappaB activation induced by phorbol ester and lipopolysaccharide. Oleandrin also blocked AP-1 activation induced by TNF and other agents and inhibited the TNF-induced activation of c-Jun NH2-terminal kinase. Overall, our results indicate that oleandrin inhibits activation of NF-kappaB and AP-1 and their associated kinases. This may provide a molecular basis for the ability of oleandrin to suppress inflammation and perhaps tumorigenesis. (+info)
Ouabain augments Ca(2+) transients in arterial smooth muscle without raising cytosolic Na(+).
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Ouabain and other cardiotonic steroids (CTS) inhibit Na(+) pumps and are widely believed to exert their cardiovascular effects by raising the cytosolic Na(+) concentration ([Na(+)](cyt)) and Ca(2+). This view has not been rigorously reexamined despite evidence that low-dose CTS may act without elevating [Na(+)](cyt); also, it does not explain the presence of multiple, functionally distinct isoforms of the Na(+) pump in many cells. We investigated the effects of Na(+) pump inhibition on [Na(+)](cyt) (with Na(+) binding benzofuran isophthalate) and Ca(2+) transients (with fura 2) in primary cultured arterial myocytes. Low concentrations of ouabain (3-100 nM) or human ouabain-like compound or reduced extracellular K(+) augmented hormone-evoked mobilization of stored Ca(2+) but did not increase bulk [Na(+)](cyt). Augmentation depended directly on external Na(+), but not external Ca(2+), and was inhibited by 10 mM Mg(2+) or 10 microM La(3+). Evoked Ca(2+) transients in pressurized small resistance arteries were also augmented by nanomolar ouabain and inhibited by Mg(2+). These results suggest that Na(+) enters a tiny cytosolic space between the plasmalemma (PL) and the adjacent sarcoplasmic reticulum (SR) via an Mg(2+)- and La(3+)-blockable mechanism that is activated by SR store depletion. The Na(+) and Ca(2+) concentrations within this space may be controlled by clusters of high ouabain affinity (alpha3) Na(+) pumps and Na/Ca exchangers located in PL microdomains overlying the SR. Inhibition of the alpha3 pumps by low-dose ouabain should raise the local concentrations of Na(+) and Ca(2+) and augment hormone-evoked release of Ca(2+) from SR stores. Thus the clustering of small numbers of specific PL ion transporters adjacent to the SR can regulate global Ca(2+) signaling. This mechanism may affect vascular tone and blood flow and may also influence Ca(2+) signaling in many other types of cells. (+info)
Endogenous digitalislike factor: an update.
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This issue of Hypertension Research contains the review and original articles presented at the International Symposium on Natriuretic and Digitalis-Like Factors held in Chitose, Hokkaido, Japan, on August 24, 1999. The symposium was the satellite meeting of the 9th International Conference on the Na/K-ATPase and Related ATPases, which was held in Sapporo, Hokkaido. At the symposium, it became clear that ouabain is the most promising candidate for a circulating hormone to regulate a number of physiological functions, including hypertension, and that other minor substances may also exist as endogenous digitalislike factors. Most of the symposium contributors submitted papers to this journal. I am going to summarize briefly the research history and current research results on endogenous digitalislike factors (EDLF). (+info)
Putative roles of ouabainlike compound in hypertension: revisited.
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It is clear that defective renal sodium handling plays an important role in the development of hypertension and that this abnormality could be caused by heterogeneous hereditary factors in the kidney. It is likely that sodium pump inhibitors with or without whole-body autoregulation gradually produce a rise in blood pressure in response to retained body sodium. Accumulated evidence has suggested that several sodium pump inhibitors similar to cardiotonic steroids are present in the human body. Ouabainlike compound (OLC) has been found to be increased with high sodium intake and hypervolemia, and in essential hypertension, mineralocorticoid hypertension, and pregnancy-induced hypertension. Further, blocking the action of OLC with digibind or a novel anti-ouabain agent has been observed to lower blood pressure in several models of experimental and clinical hypertension. The blockade of OLC action may become the basis of novel rational antihypertensive agents and may help to solve the problems still present in the management of hypertensive patients. (+info)
Liquid chromatography mass spectrometric analysis of ouabainlike factor in biological fluid.
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Ouabainlike factor (OLF), assayed as ouabainlike immunoreactivity (OLI), is a probable endogenous digitalislike factor (EDLF). Liquid chromatography/mass spectrometry (LC/MS) is one of the most highly sensitive tools for obtaining structural information regarding low-molecular weight materials in a target compound, and to measure the concentrations of these materials. We have previously reported that OLI can be isolated from the culture supernatant of the rat pheochromocytoma cell line, PC12, by several reverse-phase chromatography and LC/MS techniques. The present study was performed to characterize OLF from biological fluids such as plasma and culture supernatant of PC12 cells by LC/MS. The previous applications of LC/MS to OLI in plasma have been limited to structural identification at the final stages of isolation, in which the starting volume of plasma has been over 10 I. In the present study, we tried to minimize the volume of plasma, and to develop a new preclearing step to gain adequate LC/MS characterization using MS/MS analysis. The plasma was acidified, and OLI was purified by ODS column chromatography. OLI in chromatographic fractions from plasma was assayed by a sensitive enzyme-linked immunosorbent assay for ouabain. After Sep-Pak treatment and two rounds of ODS column chromatography, OLI was identified from 80 ml of plasma. The structure of the purified OLI was identical to authentic ouabain and digoxin, as assessed by LC/MS. In conclusion, we identified the chemically or structurally clarified ouabain and digoxin as the circulating form in plasma by LC/MS. (+info)
Analysis of human ouabainlike compound by micellar electrokinetic chromatography.
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In this preliminary study we have optimised micellar electrokinetic chromatography (MEKC, a form of capillary electrophoresis) to enable the chromatographic and spectral characteristics of human ouabainlike compound (OLC) to be investigated. Sera from fifty patients were combined to form a pool (100 ml) whilst urine (92.5 ml) was obtained from a normal healthy volunteer. Both samples were initially concentrated and partially purified by solid phase extraction before further purification by sequential HPLC separations. Final volumes for both extracts were 100 microl. MEKC was performed on a HP(3D) CE instrument with voltage set at 20 KV, capillary temperature at 20 degrees C, injection time 4 s, sample volume 10 nl, with detection by photodiode array. A compound was found in both serum and urine that had similar elution and spectral characteristics to authentic ouabain. We conclude that MEKC is potentially a useful tool for the analysis of human OLC. (+info)
The structure of the digitalislike and natriuretic factors identified as macrocyclic derivatives of the inorganic carbon suboxide.
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The Natriuretic and Endogenous DigitalisLike Factors (EDLFs) are disclosed to be cyclomeric and macroring closed derivatives of the inorganic carbon suboxide. The macrocyclic cyclohexamer with six carbon suboxide units has a molar mass of 408.2 Da, as previously been found for the EDLF of animal origin. The anhydrous cyclohexameric factor is lipophilic but is transformed into more hydrophilic derivatives by the stepwise addition of water. Based on the present findings, it appears that EDLFs exist in solution as an equilibrium mixture of lipophilic and hydrophilic forms and not as a single chemical substance. This structural assumption better accounts for the earlier observed highly anomalous properties of EDLFs. The simultaneously found higher molar mass (4,100 and 4,900 Da) macrocyclic carbon suboxide derivatives are tentatively identified as the Natriuretic factors. (+info)
Receptor occupancy with digoxin vs receptor occupancy with a putative endogenous digitalislike factor.
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The possibility that an endogenous ligand for the digitalis receptor might exist has been a source of speculation resulting in efforts over the past decades to identify such a hormone. In the current context it is of interest that prolonged wash of myocardial and skeletal muscular samples from subjects who were not in digoxin treatment generally resulted in small tendencies to increase 3H-ouabain binding ranging from 2 to 9% and from -2 to 7%, respectively. It may be appreciated that neither wet weight nor water content of left ventricular or skeletal muscular samples have been found to change as a result of the prolonged wash. Although these tendencies most likely are a mere play of chance, it may be argued that the studies did not entirely rule out the possibility of the existence of a quantitatively small amount of endogenous digitalislike factor. However, based on the evaluations of receptor occupancy with digoxin during treatment of 24-34% in the left ventricle and 9-13% in skeletal muscle, it would seem reasonable to expect that a comparatively larger fraction of digitalis receptors should be occupied by a putative endogenous digitalislike factor, if such a factor were to be of any physiological significance. (+info)