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Abstract: Changes in myocardial expression of Na+/K+-ATPase α-subunit isoforms have been demonstrated in different models of cardiac hypertrophy and hypertension. Here we studied the expression of these isozymes in stroke-prone spontaneously hypertensive rats (SHRSP) and the influence of high salt diet and treatment with the dihydropyridine lacidipine. Adult SHRSP were offered either 1% NaCl or water as drinking solution for 6 weeks. Salt-loaded SHRSP were treated or not with 1 mg/kg/day lacidipine. Compared to Wistar Kyoto (WKY) rats, non-salt-loaded SHRSP presented significant hypertension and cardiac hypertrophy. Salt intake markedly enhanced cardiac hypertrophy, an effect blunted by lacidipine. [3H]Ouabain binding assays on total particulate fractions from heart ventricles revealed the existence of two high-affinity sites with K d ∼25 and ∼200 nM, ascribed to the α3 and α2 isoforms, respectively. B max of α3 was unexpectedly high (40% of total high-affinity binding) in ventricles from WKY rats but very low in all groups of SHRSP. On the other hand, B max of α2 was similar in WKY and non-salt-loaded SHRSP; however, salt loading of SHRSP resulted in a B max reduction of 20% (P <0.05), an effect blocked by lacidipine. These effects were largely confirmed by immunoblotting analysis, which, in addition, demonstrated that the density of the ubiquitous α1 isoform was comparable among the experimental groups. In conclusion, WKY rats showed a high myocardial expression of the Na+/K+-ATPase α3 subunit, which was not found in SHRSP; the level of the α2 isoform was similar in untreated SHRSP and WKY; salt-loading of SHRSP promoted reduction of the α2 isoform, and this effect was completely hampered by lacidipine. [Copyright &y& Elsevier]

Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na+/K+-ATPase inhibition, they activate signal transduction via protein–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 μM (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-3β inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3β at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/β-catenin pathway by acting upstream to β-catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity. [ABSTRACT FROM AUTHOR]

The four isoforms of the catalytic subunit of Na+/K+-ATPase identified in rats differ in their affinities for ions and ouabain. Moreover, its expression is tissue-specific, developmentally and hormonally regulated. The aim of the present work was to evaluate the influence of age on the ratio and density of these isoforms in crude membrane preparations from rat brain hemispheres, brainstem, heart ventricles and kidneys. In all tissues investigated, Na+/K+-ATPase activity was higher in adults than in neonates but brain tissues presented the most remarkable differences. In these tissues, ouabain inhibition curves for Na+/K+-ATPase activity revealed the presence of two processes with different sensitivities to ouabain. An increase of approximately sixfold in the expression of the high affinity isoforms was observed between newborn and adult rats. In contrast, the low affinity isoform increased only approximately twofold in brainstem whereas it increased ninefold in brain hemispheres. Unlike brain tissues, a decrease (almost fourfold) in the number of high affinity ouabain binding sites was observed during ontogenesis of the heart. Although limited by the inability to resolve α2 and α3 isoforms, present data indicate that the influence of development on the expression of Na+/K+-ATPase depends not only on the isoform, but also on the tissue where the enzyme is expressed. [Copyright &y& Elsevier]

Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger : ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-( N -suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na + /K + -ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na + /K + -ATPase in a concentration-dependent manner, although arenobufagin (IC 50 = 28.3 nM) and bufalin (IC 50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC 50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na + /K + -ATPase. [ABSTRACT FROM AUTHOR]

Cardiotonic steroids (CS), natural compounds with traditional use in cardiology, have been recently suggested to exert potent anticancer effects. However, the repertoire of molecules with Na,K-ATPase activity and anticancer properties is limited. This paper describes the synthesis of 6 new digoxin derivatives substituted (on the C17-butenolide) with γ-benzylidene group and their cytotoxic effect on human fibroblast (WI-26 VA4) and cancer (HeLa and RKO) cell lines as well as their effect on Na,K-ATPase activity and expression. As digoxin, compound BD-4 was almost 100-fold more potent than the other derivatives for cytotoxicity with the three types of cells used and was also the only one able to fully inhibit the Na,K-ATPase of HeLa cells after 24 h treatment. No change in the Na,K-ATPase α1 isoform protein expression was detected. On the other hand it was 30–40 fold less potent for direct Na,K-ATPase inhibition, when compared to the most potent derivatives, BD-1 and BD-3, and digoxin. The data presented here demonstrated that the anticancer effect of digoxin derivatives substituted with γ-benzylidene were not related with their inhibition of Na,K-ATPase activity or alteration of its expression, suggesting that this classical molecular mechanism of CS is not involved in the cytotoxic effect of our derivatives. [ABSTRACT FROM AUTHOR]

Abstract: Aims: Cardiac glycosides have been extensively used in the treatment of congestive heart failure for more than 200years. Recently, cardenolides and bufadienolides were isolated from mammalian tissue and are considered as a new class of steroidal hormones. The aim of the present work was to characterize the interaction between the most clinical used cardiac glycoside digoxin and the cardiac glycosides known to exist endogenously, i.e., ouabain, marinobufagin and telocinobufagin, on human kidney Na+/K+-ATPase. Main methods: Inhibition of Na+/K+-ATPase activity from crude membrane preparations of human kidney was performed using increasing concentrations of the drugs alone or mixtures of ouabain:digoxin, telocinobufagin:digoxin and marinobufagin:digoxin in a fixed ratio 1:4, 2:3 and 3:2, respectively. The colorimetric method of Fiske and Subbarow was used to measure the inorganic phosphate released. Key findings: Analyses of inhibition curves showed that the experimental curves for all combinations were superimposed on the theoretical additive curves indicating that an additive effect occurs among distinct cardenolides and bufadienolides combinations on the human α1β1 Na+/K+-ATPase protomer. Significance: Considering the extensive use of digoxin in the treatment of heart failure and the recent findings that endogenous cardiac glycosides may have altered levels in many diseases, including heart failure, the demonstration of additive effect between cardiac glycosides can help in the understanding of recent clinical observations, including that lower than usual doses of cardiac glycosides are necessary for decreasing mortality in these patients. [Copyright &y& Elsevier]

Abstract: Ca2+ ions are essential to myonecrosis, a serious complication of snake envenomation, and heparin seems to counteract this effect. We investigated the effect of local injection of Bothrops jararacussu venom in mouse fast-twitch extensor digitorum longus (EDL) muscle, without or with heparin, on functional/molecular alterations of two central proteins involved in intracellular Ca2+ homeostasis, sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and Na+/K+-ATPase. EDL-specific SERCA1 isoform expression dropped significantly just after venom administration (up to 60% compared to control EDL values at days 1 and 3; p <0.05) while SERCA2 and Na+/K+-ATPase α1 isoform expression increased at the same time (3–6- and 2–3-fold, respectively; p <0.05). Although not significant, Na+/K+-ATPase α2 isoform followed the same trend. Except for SERCA2, all proteins reached basal levels at the 7th day. Intravenous heparin treatment did not affect these profiles. Ca2+-ATPase activity was also decreased during the first days after venom injection, but here heparin was effective to reinstate activity to control levels within 3 days. We also showed that B. jararacussu venom directly inhibited Ca2+-ATPase activity in a concentration-dependent manner. Our results indicate that EDL SERCA and Na+/K+-ATPase are importantly affected by B. jararacussu venom and heparin has protective effect on activity but not on protein expression. [Copyright &y& Elsevier]

Abstract: The use of combination drugs is very common in therapeutics as in the treatment of infectious diseases, cancer and heart failure but controversies about analysis of these interactions are frequent. The aim of the present work was to characterize the interaction between ouabain and 8-methoxy-3,9-dihydroxy coumestan (LQB93), a non-steroidal synthetic inhibitor of Na+,K+-ATPase, as well as the interaction between ouabain and ouabagenin, two cardiac glycosides sharing the same binding site. Inhibition of rat kidney Na+,K+-ATPase with increasing concentrations of the drugs alone or of mixtures of ouabain:ouabagenin and LQB93:ouabain in a fixed 1:4 ratio was performed. In other experiments, increasing concentrations of LQB93 (or ouabain) in the presence of a fixed concentration of ouabain (or ouabagenin) were used for determining the concentration pairs eliciting 50% inhibition in order to construct isobolograms. The mixture (experimental) curve for the ouabain:ouabagenin combination was superimposed on the additive (theoretical) curve indicating additivity, in accordance with the isobolographic analysis. On the other hand, the empirical curve for LQB93:ouabain (IC50 =10.6 μM) was significantly shifted to the left in relation to the theoretical curve (IC50 =30.7 μM) indicating synergism, further confirmed by the isobolographic analysis. As a conclusion, we show that the combination of a newly synthesized non-steroidal inhibitor and ouabain have a synergistic effect on Na+,K+-ATPase, further supporting a mechanism of inhibition different from ouabain. Present data also support the use of both the isobolograms and combination curves for the assessment of drug interactions occurring at the same molecular target, a situation poorly investigated. [Copyright &y& Elsevier]

Abstract: Coumestans 2a–i, bearing different patterns of substitution in A- and D-rings, were synthesized and evaluated as inhibitors of kidney Na+,K+-ATPase and ligands for the central benzodiazepine (BZP) receptor. The presence of a hydroxyl group in position 2 favours the effect on Na+,K+-ATPase but decreases the affinity for the BZP receptor, allowing the design of more selective molecules than the natural wedelolactone. On the other hand, the presence of a catechol in ring D is important for the effect on both molecular targets. [Copyright &y& Elsevier]

Abstract: The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+–Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+–Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30–50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+–Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+–Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+–Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens. [Copyright &y& Elsevier]

There is a renewed interest in the Na+/K+-ATPase (NKA, EC 3.6.3.9) either as a target for new therapeutic uses or for understanding the putative pathophysiological role of its mammalian endogenous ligands. Recent data indicate that bufalin binds to the pig kidney NKA in a way different from ouabain and digoxin, raising the question of a putative class difference between bufadienolides and cardenolides. The purpose of this work was to perform a study of the relationship between structure and both activity and kinetics, focusing mainly on the influence of the lactone ring in C17 (5 vs. 6 membered), the effect of C14-15 cyclization and the carbohydrate moiety in C3. We compared the potency of fourteen related cardiotonic steroids (CTS) for inhibition of the cycling pig kidney NKA in two different concentrations of K+, as well as the affinity for binding to the E2P conformation of the enzyme (Mg-Pi medium) and the potency for inhibiting the E2[2K] conformation of the NKA (K+-pNPPase activity). Cardenolides were clearly sensitive to the antagonistic effect of high K+ concentrations whereas bufadienolides were not or less sensitive. The C14-15 cyclization observed in some bufadienolides, such as resibufogenin and marinobufagin, caused a drastic fall in the affinity for binding to the NKA in the E2P conformation and increased the velocity of K+-pNPPase inhibition. The absence of a carbohydrate moiety in C3 increased the velocity of inhibition. Cardenolides were much more dependent on the E2P conformation for binding than bufadienolides since their ratios of E2[2K] IC 50 to E2P K i were higher than for bufadienolides. Therefore, the present data established the remarkable influence of C14-15 cyclization and of the carbohydrate moiety in C3 on both affinity and kinetics of CTS and indicate that, as a class, bufadienolides would harbor qualitative differences from cardenolides with respect to the NKA conformations to which they can bind. [ABSTRACT FROM AUTHOR]